Compositions and methods for treating infections using analogues of indolicidin

ABSTRACT

Compositions and methods for treating infections, especially bacterial infections, are provided. Indolicidin peptide analogues containing at least two basic amino acids are prepared. The analogues are administered as modified peptides, preferably containing photo-oxidized solubilizer.

CROSS-RELATED APPLICATIONS

This application is a continuation of allowed U.S. patent applicationSer. No. 09/667,486, filed Sep. 22, 2000 and issued as U.S. Pat. No.6,538,106 on Mar. 25, 2003, which is a continuation of U.S. patentapplication Ser. No. 08/915,314, filed Aug. 20, 1997 and issued as U.S.Pat. No. 6,180,604 on Jan. 30, 2001, which application claims priorityfrom U.S. Provisional Application No. 60/024,754, filed Aug. 21, 1996and U.S. Provisional Application No. 60/034,949, filed Jan. 13, 1997.

TECHNICAL FIELD

The present invention relates generally to treatment ofmicroorganism-caused infections, and more specifically, to compositionscomprising indolicidin analogues, polymer-modified analogues, and theiruses in treating infections.

BACKGROUND OF THE INVENTION

For most healthy individuals, infections are irritating, but notgenerally life-threatening. Many infections are successfully combated bythe immune system of the individual. Treatment is an adjunct and isgenerally readily available in developed countries. However, infectiousdiseases are a serious concern in developing countries and inimmunocompromised individuals.

In developing countries, the lack of adequate sanitation and consequentpoor hygiene provide an environment that fosters bacterial, parasitic,fungal and viral infections. Poor hygiene and nutritional deficienciesmay diminish the effectiveness of natural barriers, such as skin andmucous membranes, to invasion by infectious agents or the ability of theimmune system to clear the agents. As well, a constant onslaught ofpathogens may stress the immune system defenses of antibody productionand phagocytic cells (e.g., polymorphic neutrophils) to subnormallevels. A breakdown of host defenses can also occur due to conditionssuch as circulatory disturbances, mechanical obstruction, fatigue,smoking, excessive drinking, genetic defects, AIDS, bone marrowtransplant, cancer, and diabetes. An increasingly prevalent problem inthe world is opportunistic infections in individuals who are HIVpositive.

Although vaccines may be available to protect against some of theseorganisms, vaccinations are not always feasible, due to factors such asinadequate delivery mechanisms and economic poverty, or effective, dueto factors such as delivery too late in the infection, inability of thepatient to mount an immune response to the vaccine, or evolution of thepathogen. For other pathogenic agents, no vaccines are available. Whenprotection against infection is not possible, treatment of infection isgenerally pursued. The major weapon in the arsenal of treatments isantibiotics. While antibiotics have proved effective against manybacteria and thus saved countless lives, they are not a panacea. Theoveruse of antibiotics in certain situations has promoted the spread ofresistant bacterial strains. And of great importance, antibacterials areuseless against viral infections.

A variety of organisms make cationic (positively charged) peptides,molecules used as part of a non-specific defense mechanism againstmicroorganisms. When isolated, these peptides are toxic to a widevariety of microorganisms, including bacteria, fungi, and certainenveloped viruses. One cationic peptide found in neutrophils isindolicidin. While indolicidin acts against many pathogens, notableexceptions and varying degrees of toxicity exist.

Although cationic peptides show efficacy in vitro against a variety ofpathogenic cells including gram-positive bacteria, gram-negativebacteria, and fungi, these peptides are generally toxic to mammals wheninjected, and therapeutic indices are usually quite small. Approaches toreducing toxicity have included development of a derivative or deliverysystem that masks structural elements involved in the toxic response orthat improves the efficacy at lower doses. Other approaches underevaluation include liposomes and micellular systems to improve theclinical effects of peptides, proteins, and hydrophobic drugs, andcyclodextrins to sequester hydrophobic surfaces during administration inaqueous media. For example, attachment of polyethylene glycol (PEG)polymers, most often by modification of amino groups, improves themedicinal value of some proteins such as asparaginase and adenosinedeaminase, and increases circulatory half-lives of peptides such asinterleukins.

None of these approaches are shown to improve administration of cationicpeptides. For example, methods for the stepwise synthesis of polysorbatederivatives that can modify peptides by acylation reactions have beendeveloped, but acylation alters the charge of a modified cationicpeptide and frequently reduces or eliminates the antimicrobial activityof the compound. Thus, for delivery of cationic peptides, as well asother peptides and proteins, there is a need for a system combining theproperties of increased circulatory half-lives with the ability to forma micellular structure.

The present invention discloses analogues of indolicidin, designed tobroaden its range and effectiveness, and further provide other relatedadvantages. The present invention also provides methods and compositionsfor modifying peptides, proteins, antibiotics and the like to reducetoxicity, as well as providing other advantages.

SUMMARY OF THE INVENTION

The present invention generally provides indolicidin analogues. Inrelated aspects, an indolicidin analogue is provided, comprising up to25 amino acids and containing the formula: RXZXXZXB (SEQ ID NO: 1);BXZXXZXB (SEQ ID NO: 2) wherein at least one Z is valine; BBBXZXXZXB(SEQ ID NO: 3); BXZXXZXBBB_(n)(AA)_(n)MILBBAGS (SEQ ID NOS: 4-7);BXZXXZXBB(AA)_(n)M (SEQ ID NOS: 8-9); LBB_(n)XZ_(n)XXZ_(n)XRK (SEQ IDNOS: 10-17); LK_(n)XZXXZXRRK (SEQ ID NOS: 18-19); BBXZXXZXBBB (SEQ IDNO: 20), wherein at least two X residues are phenylalanine; BBXZXXZXBBB(SEQ ID NO: 21), wherein at least two X residues are tyrosine; andwherein Z is proline or valine; X is a hydrophobic residue; B is a basicamino acid; AA is any amino acid, and n is 0 or 1. In preferredembodiments, Z is proline, X is tryptophan and B is arginine or lysine.In other aspects, indolicidin analogues having specific sequences areprovided. In certain embodiments, the indolicidin analogues are coupledto form a branched peptide. In other embodiments, the analogue has oneor more amino acids altered to a corresponding D-amino acid, and incertain preferred embodiments, the N-terminal and/or the C-terminalamino acid is a D-amino acid. Other preferred modifications includeanalogues that are acetylated at the N-terminal amino acid, amidated atthe C-terminal amino acid, esterified at the C-terminal amino acid,modified by incorporation of homoserine/homoserine lactone at theC-terminal amino acid, and conjugated with polyethylene glycol orderivatives thereof.

In other aspects, the invention provides an isolated nucleic acidmolecule whose sequence comprises one or more coding sequences of theindolicidin analogues, expression vectors, and host cells transfected ortransformed with the expression vector.

Other aspects provide a pharmaceutical composition comprising at leastone indolicidin analogue and a physiologically acceptable buffer,optionally comprising an antibiotic agent. Preferred combinationsinclude I L K K F P F F P F R R K (SEQ ID NO: 22) and Ciprofloxacin; I LK K F P F F P F R R K (SEQ ID NO: 22) and Mupirocin; I L K K Y P Y Y P YR R K (SEQ ID NO: 23) and Mupirocin; I L K K W P W W P W R K (SEQ IDNO:24) and Mupirocin; I L R R W P W W P W R R R (SEQ ID NO: 25) andPiperacillin; W R I W K P K W R L P K W (SEQ ID NO: 26) andCiprofloxacin; W R I W K P K W R L P K W (SEQ ID NO: 26) and Mupirocin;W R I W K P K W R L P K W (SEQ ID NO: 26) and Piperacillin; I L R W V WW V W R R K (SEQ ID NO: 27) and Piperacillin; and I L K K W P W W P W K(SEQ ID NO: 28) and Mupirocin. In other embodiments, the pharmaceuticalcomposition further comprises an antiviral agent, (e.g., acyclovir;amantadine hydrochloride; didanosine; edoxudine; famciclovir; foscarnet;ganciclovir; idoxuridine; interferon; lamivudine; nevirapine;penciclovir; podophyllotoxin; ribavirin; rimantadine; sorivudine;stavudine; trifluridine; vidarabine; zalcitabine and zidovudine); anantiparasitic agent (e.g. 8-hydroxyquinoline derivatives; cinchonaalkaloids; nitroimidazole derivatives; piperazine derivatives;pyrimidine derivatives and quinoline derivatives, albendazole;atovaquone; chloroquine phosphate; diethylcarbamazine citrate;eflornithine; halofantrine; iodoquinol; ivermectin; mebendazole;mefloquine hydrochloride; melarsoprol B; metronidazole; niclosamide;nifurtimox; paromomycin; pentamidine isethionate; piperazine;praziquantel; primaquine phosphate; proguanil; pyrantel pamoate;pyrimethamine; pyrvinium pamoate; quinidine gluconate; quinine sulfate;sodium stibogluconate; suramin and thiabendazole); an antifungal agent(e.g., allylamines; imidazoles; pyrimidines and triazoles,5-fluorocytosine; amphotericin B; butoconazole; chlorphenesin;ciclopirox; clioquinol; clotrimazole; econazole; fluconazole;flucytosine; griseofulvin; itraconazole; ketoconazole; miconazole;naftifine hydrochloride; nystatin; selenium sulfide; sulconazole;terbinafine hydrochloride; terconazole; tioconazole; tolnaftate andundecylenate). In yet other embodiments, the composition is incorporatedin a liposome or a slow-release vehicle.

In yet another aspect, the invention provides a method of treating aninfection, comprising administering to a patient a therapeuticallyeffective amount of a pharmaceutical composition. The infection may becaused by, for example, a microorganism, such as a bacterium(e.g.,Gram-negative or Gram-positive bacterium or anaerobe; examples areAcinetobacter spp., Enterobacter spp., E. coli, H. influenzae, K.pneumoniae, P. aeruginosa, S. marcescens and S. maltophilia, Bordetellapertussis; Brucella spp.; Campylobacter spp.; Haemophilus ducreyi;Helicobacter pylori; Legionella spp.; Moraxella catarrhalis; Neisseriaspp.; Salmonella spp.; Shigella spp. and Yersinia spp.; E. faecalis, S.aureus, E. faecium, S. pyogenes, S. pneumoniae and coagulase-negativestaphylococci; Bacillus spp.; Corynebacterium spp.; Diphtheroids;Listeria spp. and Viridans Streptococci.; Clostridium spp., Bacteroidesspp. and Peptostreptococcus spp.; Borrelia spp.; Chlamydia spp.;Mycobacterium spp.; Mycoplasma spp.; Propionibacterium acne; Rickettsiaspp.; Treponema spp. and Ureaplasma spp.) fungus (e.g., yeast and/ormold), parasite (e.g., protozoan, nematode, cestode and trematode, suchas Babesia spp.; Balantidium coli; Blastocystis hominis; Cryptosporidiumparvum; Encephalitozoon spp.; Entamoeba spp.; Giardia lamblia;Leishmania spp.; Plasmodium spp.; Toxoplasma gondii; Trichomonas spp.Trypanosoma spp, Ascaris lumbricoides; Clonorchis sinensis; Eehinococcusspp.; Fasciola hepatic, Fasciolopsis buski; Heterophyes heterophyes;Hymenolepis spp.; Schistosoma spp.; Taenia spp. and Trichinellaspiralis) or virus (e.g., Alphavirus; Arenavirus; Bunyavirus;Coronavirus; Enterovirus; Filovirus; Flavivirus; Hantavirus; HTLV-BLV;Influenzavirus; Lentivirus; Lyssavirus; Paramyxovirus; Reovirus;Rhinovirus and Rotavirus, Adenovirus; Cytomegalovirus; Hepadnavirus;Molluscipoxvirus; Orthopoxvirus; Papillomavirus; Parvovirus;Polyomavirus; Simplexvirus and Varicellovirus).

In other aspects, a composition is provided, comprising an indolicidinanalogue and an antibiotic. In addition, a device, which may be amedical device, is provided that is coated with the indolicidin analogueand may further comprise an antibiotic agent.

In other aspects, antibodies that react specifically with any one of theanalogues described herein are provided. The antibody is preferably amonoclonal antibody or single chain antibody.

In a preferred aspect, the invention provides a composition comprising acompound modified by derivatization of an amino group with a conjugatecomprising activated polyoxyalkylene glycol and a fatty acid. Inpreferred embodiments, the conjugate further comprises sorbitan linkingthe polyoxyalkylene glycol and fatty acid, and more preferably ispolysorbate. In preferred embodiments, the fatty acid is from 12-18carbons, and the polyoxyalkylene glycol is polyoxyethylene, such as witha chain length of from 2 to 100. In certain embodiments, the compound isa peptide or protein, such as a cationic peptide (e.g., indolicidin oran indolicidin analogue). In preferred embodiments, the polyoxyalkyleneglycol is activated by irradiation with ultraviolet light.

The invention also provides a method of making a compound modified witha conjugate of an activated polyoxyalkylene glycol and a fatty acid,comprising: (a) freezing a mixture of the conjugate of an activatedpolyoxyalkylene glycol and fatty acid with the compound; and (b)lyophilizing the frozen mixture; wherein the compound has a free aminogroup. In preferred embodiments, the compound is a peptide orantibiotic. In other preferred embodiments, the mixture in step (a) isin an acetate buffer. In a related aspect, the method comprises mixingthe conjugate of an activated polyoxyalkylene glycol and fatty acid withthe compound; for a time sufficient to form modified compounds, whereinthe mixture is in a carbonate buffer having a pH greater than 8.5 andthe compound has a free amino group. The modified compound may beisolated by reversed-phase HPLC and/or precipitation from an organicsolvent.

The invention also provides a pharmaceutical composition comprising atleast one modified compound and a physiologically acceptable buffer. andin certain embodiments, further comprises an antibiotic agent, antiviralagent, an antiparasitic agent, and/or antifungal agent. The compositionmay be used to treat an infection, such as those caused by amicroorganism (e.g., bacterium, fungus, parasite and virus).

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth below whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.), and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an SDS-PAGE showing the extraction profile of inclusion bodies(ib) from whole cells containing MBI-11 fusion protein. The fusionprotein band is indicated by the arrow head. Lane 1, protein standards;lane 2, total lysate of XL1 Blue without plasmid; lane 3, total lysateof XL1 Blue (pR2h-11, pGP1-2), cultivated at 30° C.; lane 4, totallysate of XL1 Blue (pR2h-11, pGP1-2), induced at 42° C.; lane 5,insoluble fraction of inclusion bodies after Triton X100 wash; lane 6,organic extract of MBI-11 fusion protein; lane 7, concentrated materialnot soluble in organic extraction solvent.

FIG. 2 is an SDS-PAGE showing the expression profile of the MBI-11fusion protein using plasmid pPDR2h-11. Lane 1, protein standards; lane2, organic solvent extracted MBI-11; lane 3, total lysate of XL1 Blue(pPDR2h-11, pGP1-2), cultured at 30° C.; lane 4, total lysate of XL1Blue (pPDR2h-11, pGP1-2), induced at 42° C.

FIG. 3 presents time kill assay results for MBI 11CN, MBI 11F4CN and MBI11B7CN. The number of colony forming units×10⁻⁴ is plotted versus time.

FIG. 4 is a graph presenting the extent of solubility of MBI 11CNpeptide in various buffers.

FIG. 5 is a reversed phase HPLC profile of MBI 11CN in formulation C1(left graph panel) and formulation D (right graph panel).

FIG. 6 presents CD spectra of MBI 11CN and MBI 11B7CN.

FIG. 7 presents results of ANTS/DPX dye release of egg PC liposomes atvarious ratios of lipid to protein.

FIG. 8 presents graphs showing the activity of MBI 11B7CN againstmid-log cells grown in terrific broth (TB) or Luria-Bretani broth (LB).

FIG. 9 shows results of treatment of bacteria with MBI 10CN, MBI 11CN,or a control peptide alone or in combination with valinomycin.

FIG. 10 is a graph showing treatment of bacteria with MBI 11B7CN in thepresence of NaCl or Mg²⁺.

FIG. 11 is a graph presenting the in vitro amount of free MBI 11CN inplasma over time. Data is shown for peptide in formulation C1 andformulation D.

FIG. 12 is a graph presenting change in in vivo MBI 11CN levels in bloodat various times after intravenous injection.

FIG. 13 is a graph presenting change in in vivo MBI 11CN levels inplasma at various times after intraperitoneal injection.

FIG. 14 is a graph showing the number of animals surviving an MSSAinfection after intraperitoneal injection of MBI 10CN, ampicillin, orvehicle.

FIG. 15 is a graph showing the number of animals surviving an MSSAinfection after intraperitoneal injection of MBI 11CN, ampicillin, orvehicle.

FIG. 16 is a graph showing the results of in vivo testing of MBI-11A1CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 17 is a graph showing the results of in vivo testing of MBI-11E3CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 18 is a graph showing the results of in vivo testing of: MBI-11F3CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 19 is a graph showing the results of in vivo testing of MBI-11G2CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 20 is a graph showing the results of in viva testing of MBI-11CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 21 is a graph showing the results of in vivo testing of MBI-11B1CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 22 is a graph showing the results of in vivo testing of MBI-11B7CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 23 is a graph showing the results of in vivo testing of MBI-11B8CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIG. 24 is a graph showing the results of in vivo testing of MBI-11G4CNagainst S. aureus (Smith). Formulated peptide at various concentrationsis administered by ip injection one hour after infection with S. aureus(Smith) by ip injection.

FIGS. 25A and B display a graph showing the number of animals survivingan S. epidermidis infection after intravenous injection of MBI 10CN,gentamicin, or vehicle. Panel A, i.v. injection 15 min post-infection;panel B, i.v. injection 60 min post-infection.

FIG. 26 is a graph showing the number of animals surviving an MRSAinfection mice after intravenous injection of MBI 11CN, gentamicin, orvehicle.

FIG. 27 presents RP-HPLC traces analyzing samples for APS-peptideformation after treatment of activated polysorbate with a reducingagent. APS-MBI-11CN peptides are formed via lyophilization in 200 mMacetic acid-NaOH, pH 4.6, 1 mg/ml MBI 11CN, and 0.5% activatedpolysorbate 80. The stock solution of activated 2.0% polysorbate istreated with (a) no reducing agent, (b) 150 mM 2-mercaptoethanol, or (c)150 mM sodium borohydride for 1 hour immediately before use.

FIG. 28 presents RP-HPLC traces monitoring the formation of APS-MBI 11CNover time in aqueous solution. The reaction occurs in 200 mM sodiumcarbonate buffer pH 10.0, 1 mg/ml MBI 11CN, 0.5% activated polysorbate80. Aliquots are removed from the reaction vessel at the indicated timepoints and immediately analyzed by RP-HPLC.

DETAILED DESCRIPTION OF THE INVENTION

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms that areused herein.

The amino acid designations herein are set forth as either the standardone- or three-letter code. A capital letter indicates an L-form aminoacid; a small letter indicates a D-form amino acid.

As used herein, “indolicidin” refers to an antimicrobial cationicpeptide. Indolicidins may be isolated from a variety of organisms. Oneindolicidin is isolated from bovine neutrophils and is a 13 amino acidpeptide amidated at the carboxy-terminus in its native form (Selsted etal., J. Biol. Chem. 267:4292, 1992). An amino acid sequence ofindolicidin is presented in SEQ ID NO: 1.

As used herein, a “peptide analogue”, “analogue”, or “variant” ofindolicidin is at least 5 amino acids in length, has at least one basicamino acid (e.g., arginine and lysine) and has anti-microbial activity.Unless otherwise indicated, a named amino acid refers to the L-form,Basic amino acids include arginine, lysine, and derivatives. Hydrophobicresidues include tryptophan, phenylalanine, isoleucine, leucine, valine,and derivatives.

Also included within the scope of the present invention are amino acidderivatives that have been altered by chemical means, such asmethylation (e.g., α methylvaline), amidation, especially of theC-terminal amino acid by an alkylamine (e.g., ethylamine, ethanolamine,and ethylene diamine) and alteration of an amino acid side chain, suchas acylation of the ε-amino group of lysine. Other amino acids that maybe incorporated in the analogue include any of the D-amino acidscorresponding to the 20 L-amino acids commonly found in proteins, iminoamino acids, rare amino acids, such as hydroxylysine, or non-proteinamino acids, such as homoserine and ornithine. A peptide analogue mayhave none or one or more of these derivatives, and D-amino acids. Inaddition, a peptide may also be synthesized as a retro-, inverto- orretro-inverto-peptide.

A. Indolicidin Analogues

As noted above, the present invention provides indolicidin analogues.These analogues may be synthesized by chemical methods, especially usingan automated peptide synthesizer, or produced by recombinant methods.The choice of an amino acid sequence is guided by a general formulapresented herein.

1. Peptide Characteristics

The present invention provides indolicidin analogues. The analogues areat least 5 or 7 amino acids in length and preferably not more than 15,20, 25, 27, 30, or 35 amino acids. Analogues from 9 to 14 residues arepreferred.

General formulas for peptide analogues in the scope of the presentinvention may be set forth as:

RXZXXZXB (SEQ ID NO: 1) (1) BXZXXZXB (SEQ ID NO: 2) (2) BBBXZXXZXB (SEQID NO: 3) (3) BXZXXZXBBB_(n)(AA)_(n)MILBBAGS (SEQ ID NOS: 4-7) (4)BXZXXZXBB(AA)_(n)M (SEQ ID NOS: 8-9) (5) LBB_(n)XZ_(n)XXZ_(n)XRK (SEQ IDNOS: 10-17) (6) LK_(n)XZXXZXRRK (SEQ ID NOS: 18-19) (7) BBXZXXZXBBB (SEQID NO: 20) (8) BBXZXXZXBBB (SEQ ID NO: 21) (9)

wherein standard single letter amino abbreviations are used and; Z isproline, glycine or a hydrophobic residue, and preferably Z is prolineor valine; X is a hydrophobic residue, such as tryptophan,phenylalanine, isoleucine, leucine and valine, and preferablytryptophan; B is a basic amino acid, preferably arginine or lysine; AAis any amino acid, and n is 0 or 1. In formula (2), at least one Z isvaline; in formula (8), at least two Xs are phenylalanine; and informula (9), at least two Xs are tyrosine. Additional residues may bepresent at the N-terminus, C-terminus, or both.

As described above, modification of any of the residues including the N-or C-terminus is within the scope of the invention. A preferredmodification of the C-terminus is amidation. Other modifications of theC-terminus include esterification and lactone formation. N-terminalmodifications include acetylation, acylation, alkylation, PEGylation,myristylation, and the like. Additionally, the peptide may be modifiedto form an APS-peptide as described below. The peptides may also belabeled, such as with a radioactive label, a fluorescent label, a massspectrometry tag, biotin and the like.

2. Peptide Synthesis

Peptide analogues may be synthesized by standard chemical methods,including synthesis by automated procedure. In general, peptideanalogues are synthesized based on the standard solid-phase Fmocprotection strategy with HATU as the coupling agent. The peptide iscleaved from the solid-phase resin with trifluoroacetic acid containingappropriate scavengers, which also deprotects side chain functionalgroups. Crude peptide is further purified using preparativereversed-phase chromatography. Other purification methods, such aspartition chromatography, gel filtration, gel electrophoresis, orion-exchange chromatography may be used.

Other synthesis techniques, known in the art, such as the tBocprotection strategy, or use of different coupling reagents or the likecan be employed to produce equivalent peptides.

Peptides may be synthesized as a linear molecule or as branchedmolecules. Branched peptides typically contain a core peptide thatprovides a number of attachment points for additional peptides. Lysineis most commonly used for the core peptide because it has one carboxylfunctional group and two (alpha and epsilon) amine functional groups.Other diamino acids can also be used. Preferably, either two or threelevels of geometrically branched lysines are used; these cores form atetrameric and octameric core structure, respectively (Tam, Proc. Natl.Acad. Sci. USA 85:5409, 1988). Schematically, examples of these coresare represented as shown:

The attachment points for the peptides are typically at their carboxylfunctional group to either the alpha or epsilon amine groups of thelysines. To synthesize these multimeric peptides, the solid phase resinis derivatized with the core matrix, and subsequent synthesis andcleavage from the resin follows standard procedures. The multimericpeptide is typically then purified by dialysis against 4 M guanidinehydrochloride then water, using a membrane with a pore size to retainonly multimers. The multimeric peptides may be used within the contextof this invention as for any of the linear peptides and are preferredfor use in generating antibodies to the peptides.

3. Recombinant Production of Peptides

Peptide analogues may alternatively be synthesized by recombinantproduction (see e.g., U.S. Pat. No. 5,593,866). A variety of hostsystems are suitable for production of the peptide analogues, includingbacteria (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae), insect(e.g., Sf9), and mammalian cells (e.g, CHO, COS-7). Many expressionvectors have been developed and are available for each of these hosts.Generally, bacteria cells and vectors that are functional in bacteriaare used in this invention. However, at times, it may be preferable tohave vectors that are functional in other hosts. Vectors and proceduresfor cloning and expression in E. coli are discussed herein and, forexample, in Sambrook et al. (Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1987) andin Ausubel et al. (Current Protocols in Molecular Biology, GreenePublishing Co., 1995).

A DNA sequence encoding one or more indolicidin analogues is introducedinto an expression vector appropriate for the host. In preferredembodiments, the analogue gene is cloned into a vector to create afusion protein. The fusion partner is chosen to contain an anionicregion, such that a bacterial host is protected from the toxic effect ofthe peptide. This protective region effectively neutralizes theantimicrobial effects of the peptide and also may prevent peptidedegradation by host proteases. The fusion partner (carrier protein) ofthe invention may further function to transport the fusion peptide toinclusion bodies, the periplasm, the outer membrane, or theextracellular environment. Carrier proteins suitable in the context ofthis invention specifically include, but are not limited to,glutathione-S-transferase (GST), protein A from Staphylococcus aureus,two synthetic IgG-binding domains (ZZ) of protein A, outer membraneprotein F, β-galactosidase (lacZ), and various products of bacteriophageλ and bacteriophage T7. From the teachings provided herein, it isapparent that other proteins may be used as carriers. Furthermore, theentire carrier protein need not be used, as long as the protectiveanionic region is present. To facilitate isolation of the peptidesequence, amino acids susceptible to chemical cleavage (e.g., CNBr) orenzymatic cleavage (e.g., V8 protease, trypsin) are used to bridge thepeptide and fusion partner. For expression in E. coli, the fusionpartner is preferably a normal intracellular protein that directsexpression toward inclusion body formation. In such a case, followingcleavage to release the final product, there is no requirement forrenaturation of the peptide. In the present invention, the DNA cassette,comprising fusion partner and peptide gene, may be inserted into anexpression vector, which can be a plasmid, virus or other vehicle knownin the art. Preferably, the expression vector is a plasmid that containsan inducible or constitutive promoter to facilitate the efficienttranscription of the inserted DNA sequence in the host. Transformationof the host cell with the recombinant DNA may be carried out byCa⁺⁺-mediated techniques, by electroporation, or other methods wellknown to those skilled in the art.

Briefly, a DNA fragment encoding a peptide analogue is derived from anexisting cDNA or genomic clone or synthesized. A convenient method isamplification of the gene from a single-stranded template. The templateis generally the product of an automated oligonucleotide synthesis.Amplification primers are derived from the 5′ and 3′ ends of thetemplate and typically incorporate restriction sites chosen with regardto the cloning site of the vector. If necessary, translationalinitiation and termination codons can be engineered into the primersequences. The sequence encoding the protein may be codon-optimized forexpression in the particular host. Thus, for example, if the analoguefusion protein is expressed in bacteria, codons are optimized forbacterial usage. Codon optimization is accomplished by automatedsynthesis of the entire gene or gene region, ligation of multipleoligonucleotides, mutagenesis of the native sequence, or othertechniques known to those in the art.

At minimum, the expression vector should contain a promoter sequence.However, other regulatory sequences may also be included. Such sequencesinclude an enhancer, ribosome binding site, transcription terminationsignal sequence, secretion signal sequence, origin of replication,selectable marker, and the like. The regulatory sequences areoperationally associated with one another to allow transcription andsubsequent translation. In preferred aspects, the plasmids used hereinfor expression include a promoter designed for expression of theproteins in bacteria. Suitable promoters, including both constitutiveand inducible promoters, are widely available and are well known in theart. Commonly used promoters for expression in bacteria includepromoters from T7, T3, T5, and SP6 phages, and the trp, lpp, and lacoperons. Hybrid promoters (see, U.S. Pat. No. 4,551,433), such as tacand trc, may also be used.

In preferred embodiments, the vector includes a transcription terminatorsequence. A “transcription terminator region” is a sequence thatprovides a signal that terminates transcription by the polymerase thatrecognizes the selected promoter. The transcription terminator may beobtained from the fusion partner gene or from another gene, as long asit is functional in the host.

Within a preferred embodiment, the vector is capable of replication inbacterial cells. Thus, the vector may contain a bacterial origin ofreplication. Preferred bacterial origins of replication include f1-oriand col E1 ori, especially the ori derived from pUC plasmids. Low copynumber vectors (e.g., pPD100) may also be used, especially when theproduct is deleterious to the host.

The plasmids also preferably include at least one selectable marker thatis functional in the host. A selectable marker gene confers a phenotypeon the host that allows transformed cells to be identified and/orselectively grown. Suitable selectable marker genes for bacterial hostsinclude the chloroamphenicol resistance gene (Cm^(r)), ampicillinresistance gene (Amp^(r)), tetracycline resistance gene (Tc^(r))kanamycin resistance gene (Kan^(r)), and others known in the art. Tofunction in selection, some markers may require a complementarydeficiency in the host.

In some aspects, the sequence of nucleotides encoding the peptideanalogue also encodes a secretion signal, such that the resultingpeptide is synthesized as a precursor protein, which is subsequentlyprocessed and secreted. The resulting secreted protein may be recoveredfrom the periplasmic space or the fermentation medium. Sequences ofsecretion signals suitable for use are widely available and are wellknown (von Heijne, J. Mol. Biol. 184:99-105, 1985).

The vector may also contain a gene coding for a repressor protein, whichis capable of repressing the transcription of a promoter that contains arepressor binding site. Altering the physiological conditions of thecell can depress the promoter. For example, a molecule may be added thatcompetitively binds the repressor, or the temperature of the growthmedia may be altered. Repressor proteins include, but are not limited tothe E. coli lacI repressor (responsive to induction by IPTG), thetemperature sensitive λcI857 repressor, and the like.

Examples of plasmids for expression in bacteria include the pETexpression vectors pET3a, pET 11a, pET 12a-c, and pET 15b (see U.S. Pat.No. 4,952,496; available from Novagen, Madison, Wis.). Low copy numbervectors (e.g., pPD100) can be used for efficient overproduction ofpeptides deleterious to the E. coli host (Dersch et al., FEMS Microbiol.Lett. 123: 19, 1994).

Bacterial hosts for the T7 expression vectors may contain chromosomalcopies of DNA encoding T7 RNA polymerase operably linked to an induciblepromoter (e.g., lacUV promoter; see, U.S. Pat. No. 4,952,496), such asfound in the E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS,HMS174(DE3) and BL21(DE3). T7 RNA polymerase can also be present onplasmids compatible with the T7 expression vector. The polymerase may beunder control of a lambda promoter and repressor (e.g., pGP1-2; Taborand Richardson, Proc. Natl. Acad. Sci. USA 82: 1074, 1985).

The peptide analogue protein is isolated by standard techniques, such asaffinity, size exclusion, or ionic exchange chromatography, HPLC and thelike. An isolated peptide should preferably show a major band byCoomassie blue stain of SDS-PAGE that is at least 90% of the material.

4. Generation of Analogues by Amplification-based Semi-randomMutagenesis

Indolicidin analogues can be generated using an amplification (e.g.,PCR)-based procedure in which primers are designed to target sequencesat the 5′ and 3′ ends of an encoded parent peptide, for exampleindolicidin. Amplification conditions are chosen to facilitatemisincorporation of nucleotides by the thermostable polymerase duringsynthesis. Thus, random mutations are introduced in the originalsequence, some of which result in amino acid alteration(s).Amplification products may be cloned into a coat protein of a phagevector, such as a phagemid vector, packaged and amplified in anacceptable host to produce a display library.

These libraries can then be assayed for antibiotic activity of thepeptides. Briefly, bacteria infected with the library are plated, grown,and overlaid with agarose containing a bacterial strain that the phageare unable to infect. Zones of growth inhibition in the agarose overlayare observed in the area of phage expressing an analogue withanti-bacterial activity. These inhibiting phage are isolated and thecloned peptide sequence determined by DNA sequence analysis. The peptidecan then be independently synthesized and its antibiotic activityfurther investigated.

5. Antibodies to Indolicidin Analogues

Antibodies are typically generated to a specific peptide analogue usingmultiple antigenic peptides (MAPs) that contain approximately eightcopies of the peptide linked to a small non-immunogenic peptidyl core toform an immunogen. (See, in general, Harlow and Lane, supra.) The MAPsare injected subcutaneously into rabbits or into mice or other rodents,where they may have sufficiently long half-lives to facilitate antibodyproduction. After twelve weeks blood samples are taken, serum isseparated and tested in an ELISA assay against the original peptide,with a positive result indicating the presence of antibodies specific tothe target peptide. This serum can then be stored and used in ELISAassays to specifically measure the amount of the specific analogue.Alternatively, other standard methods of antibody production may beemployed, for example generation of monoclonal antibodies.

Within the context of the present invention, antibodies are understoodto include monoclonal antibodies, polyclonal antibodies, anti-idiotypicantibodies, antibody fragments (e.g., Fab, and F(ab′)₂, F_(v) variableregions, or complementarity determining regions). Antibodies aregenerally accepted as specific against indolicidin analogues if theybind with a K_(d) of greater than or equal to 10⁻⁷M, preferably greaterthan of equal to 10⁻⁸M. The affinity of a monoclonal antibody or bindingpartner can be readily determined by one of ordinary skill in the art(see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949). Once suitableantibodies have been obtained, they may be isolated or purified by manytechniques well known to those of ordinary skill in the art.

Monoclonal antibodies may also be readily generated from hybridoma celllines using conventional techniques (see U.S. Pat. Nos. RE 32,011,4,902,614, 4,543,439, and 4,411,993; see also Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988). Briefly, within one embodiment, a subject animal such as a rat ormouse is injected with peptide, generally administered as an emulsion inan adjuvant such as Freund's complete or incomplete adjuvant in order toincrease the immune response. The animal is generally boosted at leastonce prior to harvest of spleen and/or lymph nodes and immortalizationof those cells. Various immortalization techniques, such as mediated byEpstein-Barr virus or fusion to produce a hybridoma, may be used. In apreferred embodiment, immortilization occurs by fusion with a suitablemyeloma cell line to create a hybridoma that secretes monoclonalantibody. Suitable myeloma lines include, for example, NS-1 (ATCC No.TIB 18), and P3X63-Ag 8.653 (ATCC No. CRL 1580). The preferred fusionpartners do not express endogenous antibody genes. After about sevendays, the hybridomas may be screened for the presence of antibodies thatare reactive against a telomerase protein. A wide variety of assays maybe utilized (see Antibodies: A Laboratory Manual, Harlow and Lane(eds.), Cold Spring Harbor Laboratory Press, 1988).

Other techniques may also be utilized to construct monoclonal antibodies(see Huse et al., Science 246:1275-1281, 1989; Sastry et al., Proc.Natl. Acad. Sci. USA 86:5728-5732, 1989; Alting-Mees et al., Strategiesin Molecular Biology 3:1-9, 1990; describing recombinant techniques).These techniques include cloning heavy and light chain immunoglobulincDNA in suitable vectors, such as λImmunoZap(H) and λImmunoZap(L). Theserecombinants may be screened individually or co-expressed to form Fabfragments or antibodies (see Huse et al., supra; Sastry et al., supra).Positive plaques may subsequently be converted to a non-lytic plasmidthat allows high level expression of monoclonal antibody fragments fromE. coli.

Similarly, portions or fragments, such as Fab and Fv fragments, ofantibodies may also be constructed utilizing conventional enzymaticdigestion or recombinant DNA techniques to yield isolated variableregions of an antibody. Within one embodiment, the genes which encodethe variable region from a hybridoma producing a monoclonal antibody ofinterest are amplified using nucleotide primers for the variable region.In addition, techniques may be utilized to change a “murine” antibody toa “human” antibody, without altering the binding specificity of theantibody.

B. Testing

Indolicidin analogues of the present invention are assessed either aloneor in combination with an antibiotic agent or another analogue for theirpotential as antibiotic therapeutic agents using a series of assays.Preferably, all peptides are initially assessed in vitro, the mostpromising candidates selected for further assessment in vivo, and usingthe results of these assays candidates are selected for pre-clinicalstudies. The in vitro assays include measurement of antibiotic activity,toxicity, solubility, pharmacology, secondary structure, liposomepermeabilization and the like. In vivo assays include assessment ofefficacy in animal models, antigenicity, toxicity, and the like. Ingeneral, in vitro assays are initially performed, followed by in vivoassays.

1. In vitro Assays

Indolicidin analogues are assessed for antibiotic activity by an assaysuch as an agarose dilution MIC assay or a broth dilution or time-killassay. Antibiotic activity is measured as inhibition of growth orkilling of a microorganism (e.g., bacteria, fungi). Briefly, a candidateanalogue in Mueller Hinton broth supplemented with calcium and magnesiumis mixed with molten agarose. Other formulations of broths and agars maybe used as long as the peptide analogue can freely diffuse through themedium. The agarose is poured into petri dishes or wells, allowed tosolidify, and a test strain is applied to the agarose plate. The teststrain is chosen, in part, on the intended application of the analogue.Thus, by way of example, if an analogue with activity against S. aureusis desired, an S. aureus strain is used. It may be desirable to assaythe analogue on several strains and/or on clinical isolates of the testspecies. Plates are incubated overnight and, on the following day,inspected visually for bacterial growth. The minimum inhibitoryconcentration (MIC) of an analogue is the lowest concentration ofpeptide that completely inhibits growth of the organism. Analogues thatexhibit good activity against the test strain, or group of strains,typically having an MIC of less than or equal to 16 μg/ml are selectedfor further testing.

The selected analogues may be further tested for their toxicity tonormal mammalian cells. An exemplary assay is a red blood cell (RBC)(erythrocyte) hemolysis assay. Briefly, red blood cells are isolatedfrom whole blood, typically by centrifugation, and washed free of plasmacomponents. A 1% (v/v) suspension of erythrocytes in isotonic saline isincubated with different concentrations of peptide analogue. Generally,the analogue will be in a suitable formulation buffer. After incubationfor approximately 1 hour at 37° C., the cells are centrifuged, and theabsorbance of the supernatant at 540 nm is determined. A relativemeasure of lysis is determined by comparison to absorbance aftercomplete lysis of erythrocytes using NH₄Cl or equivalent (establishing a100% value). An analogue that is not lytic, or is only moderately lytic,as exemplified in Example 8, is desirable and is suitable for furtherscreening. Other in vitro toxicity assays, for example measurement oftoxicity towards cultured mammalian cells, may be used to assess invitro toxicity.

Solubility of the peptide analogue in formulation buffer is anadditional parameter that may be examined. Several different assays maybe used, such as appearance in buffer. Briefly, peptide analogue issuspended in solution, such as broth or formulation buffer. Theappearance is evaluated according to a scale that ranges from (a) clear,no precipitate, (b) light, diffuse precipitate, to (c) cloudy, heavyprecipitate. Finer gradations may be used. In general, less precipitateis more desirable. However, some precipitate may be acceptable.

Additional in vitro assays may be carried out to assess the potential ofthe analogue as a therapeutic. Such assays include peptide solubility informulations, pharmacology in blood or plasma, serum protein binding,analysis of secondary structure, for example by circular dichroism,liposome permeabilization, and bacterial inner membranepermeabilization. In general, it is desirable that analogues are solubleand perform better than indolicidin.

2. In vivo Assays

Analogues selected on the basis of the results from the in vitro assayscan be tested in vivo for efficacy, toxicity and the like.

The antibiotic activity of selected analogues may be assessed in vivofor their ability to ameliorate microbial infections using animalmodels. Within these assays, an analogue is useful as a therapeutic ifinhibition of microorganismal growth compared to inhibition with vehiclealone is statistically significant. This measurement can be madedirectly from cultures isolated from body fluids or sites, orindirectly, by assessing survival rates of infected animals. Forassessment of antibacterial activity several animal models areavailable, such as acute infection models including those in which (a)normal mice receive a lethal dose of microorganisms, (b) neutropenicmice receive a lethal dose of microorganisms or (c) rabbits receive aninoculum in the heart, and chronic infection models. The model selectedwill depend in part on the intended clinical indication of the analogue.

By way of example, in one such normal mouse model, mice are inoculatedip or iv with a lethal dose of bacteria. Typically, the dose is suchthat 90-100% of animals die within 2 days. The choice of amicrorganismal strain for this assay depends, in part, upon the intendedapplication of the analogue, and in the accompanying examples, assaysare carried out with three different Staphylococcus strains. Briefly,shortly before or after inoculation (generally within 60 minutes),analogue in a suitable formulation buffer is injected. Multipleinjections of analogue may be administered. Animals are observed for upto 8 days post-infection and the survival of animals is recorded.Successful treatment either rescues animals from death or delays deathto a statistically significant level, as compared with non-treatmentcontrol animals. Analogues that show better efficacy than indolicidinitself are preferred.

In vivo toxicity of an analogue is measured through administration of arange of doses to animals, typically mice, by a route defined in part bythe intended clinical use. The survival of the animals is recorded andLD₅₀, LD₉₀₋₁₀₀, and maximum tolerated dose (MTD) can be calculated toenable comparison of analogues. Analogues less toxic than indolicidinare preferred.

Additional in vivo assays may be performed to assist in the selection ofanalogues for clinical development. For example, immunogenicity ofanalogues can be evaluated, typically by injection of the analogue informulation buffer into normal animals, generally mice, rats, orrabbits. At various times after injection, serum is obtained and testedfor the presence of antibodies that bind to the analogue. Testing aftermultiple injections, mimicking treatment protocols, may also beperformed. Antibodies to analogues can be identified by ELISA,immunoprecipitation assays, Western blots, and other methods. (see,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1988). Analogues that elicitno or minimal production of antibodies are preferred. Additionally,pharmacokinetics of the analogues in animals and histopathology ofanimals treated with analogues may be determined.

Selection of indolicidin analogues as potential therapeutics is based onin vitro and in vivo assay results. In general, peptide analogues thatexhibit low toxicity at high dose levels and high efficacy at low doselevels are preferred candidates.

3. Synergy Assays

For assessment of analogues in combination with an antibiotic or anotheranalogue, the combination can be subjected to the above series ofassays. Antibiotics include any chemical that tends to prevent, inhibitor destroy life and as such, antibiotics include anti-bacterial agents,anti-fungicides, anti-viral agents, and anti-parasitic agents. Merely byway of example, anti-bacterial antibiotics are discussed. Methods formixing and administering the components vary depending on the intendedclinical use of the combination.

Briefly, one assay for in vitro anti-bacterial activity, the agarosedilution assay, is set up with an array of plates that each contain acombination of peptide analogue and antibiotic in variousconcentrations. The plates are inoculated with bacterial isolates,incubated, and the MICs of the components recorded. These results arethen used to calculate the FIC. Antibiotics used in testing include, butare not limited to, penicillins, cephalosporins, carbacephems,cephamycins, carbapenems, monobactams, aminoglycosides, glycopeptides,quinolones, tetracyclines, macrolides, and fluoroquinolones (see Table 1below).

Examples of antibiotics, include, but are not limited to, Penicillin G(CAS Registry No.: 61-33-6); Methicillin (CAS Registry No.: 61-32-5);Nafcillin (CAS Registry No.: 147-52-4); Oxacillin (CAS Registry No.:66-79-5); Cloxacillin (CAS Registry No.: 61-72-3); Dicloxacillin (CASRegistry No.: 3116-76-5); Ampicillin (CAS Registry No.: 69-53-4);Amoxicillin (CAS Registry No.: 26787-78-0); Ticarcillin (CAS RegistryNo.: 34787-01-4); Carbenicillin (CAS Registry No.: 4697-36-3);Mezlocillin (CAS Registry No.: 51481-65-3); Azlocillin (CAS RegistryNo.: 37091-66-0); Piperacillin (CAS Registry No.: 61477-96-1); Imipenem(CAS Registry No.: 74431-23-5); Aztreonam (CAS Registry No.:78110-38-0); Cephalothin (CAS Registry No.: 153-61-7); Cefazolin (CASRegistry No.: 25953-19-9); Cefaclor (CAS Registry No.: 70356-03-5);Cefamandole formate sodium (CAS Registry No.: 42540-40-9); Cefoxitin(CAS Registry No.: 35607-66-0); Cefuroxime (CAS Registry No.:55268-75-2); Cefonicid (CAS Registry No.: 61270-58-4); Cefmetazole (CASRegistry No.: 56796-20-4); Cefotetan (CAS Registry No.: 69712-56-7);Cefprozil (CAS Registry No.: 92665-29-7); Loracarbef (CAS Registry No.:121961-22-6); Cefetamet (CAS Registry No.: 65052-63-3); Cefoperazone(CAS Registry No.: 62893-19-0); Cefotaxime (CAS Registry No.:63527-52-6); Ceftizoxime (CAS Registry No.: 68401-81-0); Ceftriaxone(CAS Registry No.: 73384-59-5); Ceftazidime (CAS Registry No.:72558-82-8); Cefepime (CAS Registry No.: 88040-23-7); Cefixime (CASRegistry No.: 79350-37-1); Cefpodoxime (CAS Registry No.: 80210-62-4);Cefsulodin (CAS Registry No.: 62587-73-9); Fleroxacin (CAS Registry No.:79660-72-3); Nalidixic acid (CAS Registry No.: 389-08-2); Norfloxacin(CAS Registry No.: 70458-96-7); Ciprofloxacin (CAS Registry No.:85721-33-1); Ofloxacin (CAS Registry No.: 82419-36-1); Enoxacin (CASRegistry No.: 74011-58-8); Lomefloxacin (CAS Registry No.: 98079-51-7);Cinoxacin (CAS Registry No.: 28657-80-9); Doxycycline (CAS Registry No.:564-25-0); Minocycline (CAS Registry No.: 10118-90-8); Tetracycline (CASRegistry No.: 60-54-8); Amikacin (CAS Registry No.: 37517-28-5);Gentamicin (CAS Registry No.: 1403-66-3); Kanamycin (CAS Registry No.:8063-07-8); Netilmicin (CAS Registry No.: 56391-56-1); Tobramycin (CASRegistry No.: 32986-56-4); Streptomycin (CAS Registry No.: 57-92-1);Azithromycin (CAS Registry No.: 83905-01-5); Clarithromycin (CASRegistry No.: 81103-11-9); Erythromycin (CAS Registry No.: 114-07-8);Erythromycin estolate (CAS Registry No.: 3521-62-8); Erythromycin ethylsuccinate (CAS Registry No.: 41342-53-4); Erythromycin glucoheptonate(CAS Registry No.: 23067-13-2); Erythromycin lactobionate (CAS RegistryNo.: 3847-29-8); Erythromycin stearate (CAS Registry No.: 643-22-1);Vancomycin (CAS Registry No.: 1404-90-6); Teicoplanin (CAS Registry No.:61036-64-4); Chloramphenicol (CAS Registry No.: 56-75-7); Clindamycin(CAS Registry No.: 18323-44-9); Trimethoprim (CAS Registry No.:738-70-5); Sulfamethoxazole (CAS Registry No.: 723-46-6); Nitrofurantoin(CAS Registry No.: 67-20-9); Rifampin (CAS Registry No.: 13292-46-1);Mupirocin (CAS Registry No.: 12650-69-0); Metronidazole (CAS RegistryNo.: 443-48-1); Cephalexin (CAS Registry No.: 15686-71-2); Roxithromycin(CAS Registry No.: 80214-83-1); Co-amoxiclavuanate; combinations ofPiperacillin and Tazobactam; and their various salts, acids, bases, andother derivatives.

TABLE 1 Class of Antibiotic Antibiotic Mode of Action PENICILLINS Blocksthe formation of new cell walls in bacteria Natural Penicillin G,Benzylpenicillin Penicillin V, Phenoxymethylpenicillin Penicillinaseresistant Methicillin, Nafcillin, Oxacillin Cloxacillin, DicloxacillinAcylamino-penicillins Ampicillin, Amoxicillin Carboxy-penicillinsTicarcillin, Carbenicillin Ureido-penicillins Mezlocillin, Azlocillin,Piperacillin CARBAPENEMS Imipenem, Meropenem Blocks the formation of newcell walls in bacteria MONOBACTAMS Blocks the formation of new cellwalls in bacteria Aztreonam CEPHALOSPORINS Prevents formation of newcell walls in bacteria 1st Generation Cephalothin, Cefazolin 2ndGeneration Cefaclor, Cefamandole Cefuroxime, Cefonicid, Cefmetazole,Cefotetan, Cefprozil 3rd Generation Cefetamet, Cefoperazone Cefotaxime,Ceftizoxime Ceftriaxone, Ceftazidime Cefixime, Cefpodoxime, Cefsulodin4th Generation Cefepime CARBACEPHEMS Loracarbef Prevents formation ofnew cell walls in bacteria CEPHAMYCINS Cefoxitin Prevents formation ofnew cell walls in bacteria QUINOLONES Fleroxacin, Nalidixic AcidInhibits bacterial DNA Norfloxacin, Ciprofloxacin synthesis Ofloxacin,Enoxacin Lomefloxacin, Cinoxacin TETRACYCLINES Doxycycline, Minocycline,Inhibits bacterial protein Tetracycline synthesis, binds to 30S ribosomesubunit. AMINOGLYCOSIDES Amikacin, Gentamicin, Kanamycin, Inhibitsbacterial protein Netilmicin, Tobramycin, synthesis, binds to 30SStreptomycin ribosome subunit. MACROLIDES Azitbromycin, Clarithromycin,inhibits bacterial protein Erythromycin synthesis, binds to 50S ribosomesubunit Derivatives of Erythromycin estolate, Erythromycin Erythromycinstearate Erytbromycin ethylsuccinate Erythromycin gluceptateErythromycin lactobionate GLYCOPEPTIDES Vancomycin, Teicoplanin Inhibitscell wall synthesis, prevents peptidoglycan elongation. MISCELLANEOUSChloramphenicol Inhibits bacterial protein synthesis, binds to 50Sribosome subunit. Clindamycin Inhibits bacterial protein synthesis,binds to 50S ribosome subunit. Trimethoprim Inhibits the enzymedihydrofolate reductase, which activates folic acid. SulfamethoxazoleActs as antimetabolite of PABA & inhibits synthesis of folic acidNitrofurantoin Action unknown, but is concentrated in urine where it canact on urinary tract bacteria Rifampin Inhibits bacterial RNA polymeraseMupirocin Inhibits bacterial protein synthesis

Synergy is calculated according to the formula below. An FIC of ≦0.5 isevidence of synergy, although combinations with higher values may betherapeutically useful.

${\frac{{MIC}\left( {{peptide}\mspace{14mu}{in}\mspace{14mu}{combination}} \right)}{{MIC}\left( {{peptide}\mspace{14mu}{alone}} \right)} + \frac{{MIC}\left( {{antibiotic}\mspace{14mu}{in}\mspace{14mu}{combination}} \right)}{{MIC}\left( {{antibiotic}\mspace{14mu}{alone}} \right)}} = {FIC}$

For example, antibiotics from the groups of penicillins, cephalosporins,carbacephems, cephamycins, carbapenems, monobactams, aminoglycosides,glycopeptides, quinolones, tetracyclines, macrolides, fluoroquinolones,and other miscellaneous antibiotics may be used in combination with anyof the peptides disclosed herein. For example, MBI 11A1CN or MBI 11D18CNwith Ciprofloxacin, MBI 11A1CN, MBI 11A3CN, MBI 11B4CN, MBI 11D18CN orMBI 11G13CN with Mupirocin, MBI 11B9CN, MBI 11D18CN or MBI 11F4CN withPiperacillin are preferred combinations.

C. Polymer Modification of Peptides and Proteins

As noted herein, the present invention provides methods and compositionsfor modifying a compound with a free amine group, such as peptides,proteins, certain antibiotics, and the like, with an activatedpolysorbate ester and derivatives. When the compounds are peptides orproteins, the modified or derivatized forms are referred to herein as“APS-modified peptides” or “APS-modified proteins”. Similarly, modifiedforms of antibiotics are referred to as “APS-modified antibiotics.”APS-modified compounds (e.g., APS-cationic peptides) have improvedpharmacological properties.

In addition to peptides and proteins, antibiotics, antifungals,anti-rythmic drugs, and any other compound with a free primary or otheramine are suitable for modification. For example, cephalosporins,aminopenicillins, ethambutol, pyrazinamide, sulfonamines, quinolones(e.g., ciprofiloxacin, clinafloxacin) aminoglycosides andspectinomhycins, including, but not limited to, streptomycin, neomycin,kanamycin, gentamicin, have free amines for modification. Anti-fungalssuch as amphotericin B, nystatin, 5-fluorocytosine, and the like haveamines available for derivativization. Anti-virals, such as tricyclicamines (e.g., amantadine); and anti-parasitic agents (e.g.,dapsone), mayall be derivatized. For exemplary purposes only, the discussion hereinis directed to modified peptides and proteins.

1. Characteristics of Reagent

As discussed herein, a suitable reagent for formation of APS-modifiedcompounds (e.g., peptides and proteins) comprises a hydrophobic regionand a hydrophilic region, and optionally a linker. The hydrophobicregion is a lipophilic compound with a suitable functional group forconjugation to the hydrophilic region or linker. The hydrophilic regionis a polyalkylene glycol. As used herein, “polyalkylene glycol” refersto 2 or 3 carbon polymers of glycols. Two carbon polyalkylenes includepolyethylene glycol (PEG) of various molecular weights, and itsderivatives, such as polysorbate. Three carbon polyalkylenes includepolypropylene glycol and its derivatives.

The hydrophobic region is generally a fatty acid, but may be a fattyalcohol, fatty thiol, and the like, which are also lipophilic compounds.The fatty acid may be saturated or unsaturated. The chain length doesnot appear to be important, although typically commercially availablefatty acids are used and have chain lengths of C₁₂₋₁₈. The length may belimited however by solubility or solidity of the compound, that islonger lengths of fatty acids are solid at room temperature. Fatty acidsof 12 carbons (lauryl), 14 carbons, 16 carbons (palmitate), and 18carbons (monostearate or oleate) are preferred chain lengths.

The hydrophilic region is a polyalkylene glycol, either polyethylene orpolypropylene glycol monoether. The ether function is formed by thelinkage between the polyoxyethylene chain, preferably having a chainlength of from 2 to 100 monomeric units, and the sorbitan group.Polymethylene glycol is unsuitable for administration in animals due toformation of formaldehydes, and glycols with a chain length of ≧4 may beinsoluble. Mixed polyoxyethylene-polyoxypropylene chains are alsosuitable.

A linker for bridging the hydrophilic and hydrophobic regions is notrequired, but if used, should be a bifunctional nucleophile able toreact with both polyalkylene glycol and the hydrophobic region. Thelinker provides electrons for a nucleophilic reaction with thepolyalkylene glycol, typically formed by reaction with ethylene oxide orpropylene oxide. Suitable linkers include sorbitan, sugar alcohols,ethanolamine, ethanolthiol, 2-mercaptoethanol, 1,6 diaminohexane, anamino acid (e.g., glutamine, lysine), other reduced sugars, and thelike. For example, sorbitan forms an ester linkage with the fatty acidin a polysorbate.

Suitable compounds include polyoxyethylenesorbitans, such as themonolaurate, monooleate, monopalmitate, monostearate, trioleate, andtristearate esters. These and other suitable compounds may besynthesized by standard chemical methods or obtained commercially (e.g.,Sigma Chemical Co., MO; Aldrich Chemical Co., WI; J. B. Baker, N.J.).

2. Activation of Reagent

The reagent, generally a polysorbate, is activated by exposure to UVlight with free exchange of air. Activation is achieved using a lampthat irradiates at 254 nm or 302 nm. Preferably, the output is centeredat 254 nm. Longer wave lengths may require longer activation time. Whilesome evidence exists that fluorescent room light can activate thepolysorbates, experiments have shown that use of UV light at 254 nmyields maximal activation before room light yields a detectable level ofactivation.

Air plays an important role in the activation of the polysorbates.Access to air doubles the rate of activation relative to activationsperformed in sealed containers. It is not yet known which gas isresponsible; an oxygen derivative is likely, although peroxides are notinvolved. UV exposure of compounds with ether linkages is known togenerate peroxides, which can be detected and quantified using peroxidetest strips. In a reaction, hydrogen peroxide at 1 to 10 fold higherlevel than found in UV-activated material was added to a polysorbatesolution in the absence of light. No activation was obtained.

The reagent is placed in a suitable vessel for irradiation. Aconsideration for the vessel is the ability to achieve uniformirradiation. Thus, if the pathlength is long, the reagent may be mixedor agitated. The activation requires air; peroxides are not involved inthe activation. The reagent can be activated in any aqueous solution andbuffering is not required.

An exemplary activation takes place in a cuvette with a 1 cm liquidthickness. The reagent is irradiated at a distance of less than 9 cm at1500 μW/cm² (initial source output) for approximately 24 hours. Underthese conditions, the activated reagent converts a minimum of 85% of thepeptide to APS-peptide.

3. Modification of Peptides or Proteins with Activated Reagent

The peptides or proteins are reacted with the APS reagent in either aliquid or solid phase and become modified by the attachment of the APSderivative. The methods described herein for attachment offer theadvantage of maintaining the charge on the peptide or protein. When thecharge of the peptide is critical to its function, such as theantibiotic activity of cationic peptides described herein, theseattachment methods offer additional advantages. Methods that attachgroups via acylation result in the loss of positive charge viaconversion of amino to amido groups. In addition, no bulky orpotentially antigenic linker, such as a triazine group, is known to beintroduced by the methods described herein.

As noted above, APS-peptide formation occurs in solid phase or inaqueous solution. Briefly, in the solid phase method, the peptide issuspended in a suitable buffer, such as an acetate buffer. Othersuitable buffers that support APS-peptide formation may also be used.The acetate buffer may be sodium, rubidium, lithium, and the like. Otheracetate solutions, such as HAc or HAc-NaOH, are also suitable. Apreferred pH range for the buffer is from 2 to 8.3, although a widerrange may be used. When the starting pH of the acetic acid-NaOH bufferis varied, subsequent lyophilization from 200 mM acetic acid bufferyields only the Type I modified peptide (see Example 14). The presenceof an alkaline buffer component results in the formation of Type IImodified peptides. A typical peptide concentration is 1 mg/ml, whichresults in 85-95% modified peptide, however other concentrations aresuitable. The major consideration for determining concentration appearsto be economic. The activated polymer (APS) is added in molar excess tothe peptide, such that a 1:1 molar ratio of APS-modified peptide isgenerated. Generally, a starting ratio of approximately 2.5:1(APS:peptide) to 5:1 (APS: peptide) yields a 1:1 APS-modified peptide.

The reaction mix is then frozen (e.g., −80° C.) and lyophilized. Sodiumacetate disproportionates into acetic acid and NaOH duringlyophilization; removal of the volatile acetic acid by the vacuum leavesNaOH dispersed throughout the result solid matrix. This loss of aceticacid is confirmed by a pH increase detected upon dissolution of thelyophilizate. No APS-modified peptide is formed in acetate buffer if thesamples are only frozen then thawed.

The modification reaction can also take place in aqueous solution.However, APS modifications do not occur at ambient temperature in anyacetate buffer system tested regardless of pH. APS modifications alsoare not formed in phosphate buffers as high as pH 11.5. APS modificationdoes occur in a sodium carbonate buffer at a pH greater than about 8.5.Other buffers may also be used if they support derivitization. A pHrange of 9-11 is also suitable, and pH 10 is most commonly used. Thereaction occurs in two phases: Type I peptides form first, followed byformation of Type II peptides.

In the present invention, linkage occurs at an amino group. For apeptide, linkage can occur at the α-NH₂ of the N-terminal amino acid orε-NH₂ group of lysine. Other primary and secondary amines may also bemodified. Complete blocking of all amino groups by acylation (MBI11CN-Y1) inhibits APS-peptide formation. Thus, modification of arginineor tryptophan residues does not occur. If the only amino group availableis the α-amino group (e.g., MBI 11B9CN and MBI 11G14CN), the Type I formis observed. The inclusion of a single lysine (e.g., MBI 11B1CN, MBI11B7CN, MBI 11B8CN), providing an ε-amino group, results in Type IIforms as well. The amount of Type II formed increases for peptides withmore lysine residues.

4. Purification and Physical Properties of APS-modified Peptides

The APS-modified peptides may be purified. In circumstances in which thefree peptide is toxic, purification may be necessary to removeunmodified peptide and/or unreacted polysorbate. Any of a variety ofpurification methods may be used. Such methods include reversed phaseHPLC, precipitation by organic solvent to remove polysorbate, sizeexclusion chromatography, ion exchange chromatography, filtration andthe like. RP-HPLC is preferred. Procedures for these separation methodsare well known.

APS-peptide (or protein) formation results in the generation ofpeptide-containing products that are more hydrophobic that the parentpeptide. This property can be exploited to effect separation of theconjugate from free peptide by RP-HPLC. The conjugates are resolved intotwo populations based on their hydrophobicity as determined by RP-HPLC;the Type I population elutes slightly earlier than the Type IIpopulation.

The MBI 11 series of peptides have molecular weights between 1600 and2500. When run on a Superose 12 column, a size exclusion column, thesepeptides elute no earlier than the bed volume indicating a molecularmass below 20 kDa. In contrast, the APS-modified peptides elute at 50kDa, thus demonstrating a large increase in apparent molecular mass.

An increase in apparent molecular mass could enhance thepharmacokinetics of the cationic peptides because increased molecularmass reduces the rate at which peptides and proteins are removed fromblood. Micelle formation may offer additional benefits by delivering“packets” of peptide molecules to microorganisms rather than relying onthe multiple binding of single peptide molecules. In addition, theAPS-modified peptides are soluble in methylene chloride or chloroform,whereas the parent peptide is essentially insoluble. This increasedorganic solubility may significantly enhance the ability to penetratetissue barriers.

In addition, by circular dichroism (CD) studies, APS-modified peptidesare observed to have an altered 3-dimensional conformation. As shown inthe Examples, MBI 11CN and MBI 11B7CN have unordered structures inphosphate buffer or 40% aqueous trifluoroethanol (TFE) and form a β-turnconformation only upon insertion into liposomes. In contrast, CD spectrafor APS-modified MBI 11CN and APS-modified MBI 11B7CN indicate β-turnstructure in phosphate buffer.

D. Formulations and Administration

As noted above, the present invention provides methods for treating andpreventing infections by administering to a patient a therapeuticallyeffective amount of a peptide analogue of indolicidin as describedherein. Patients suitable for such treatment may be identified bywell-established hallmarks of an infection, such as fever, pus, cultureof organisms, and the like. Infections that may be treated with peptideanalogues include those caused by or due to microorganisms. Examples ofmicroorganisms include bacteria (e.g., Gram-positive, Gram-negative),fungi, (e.g., yeast and molds), parasites (e.g., protozoans, nematodes,cestodes and trematodes), viruses, and prions. Specific organisms inthese classes are well known (see for example, Davis et al.,Microbiology, 3^(rd) edition, Harper & Row, 1980). Infections include,but are not limited to, toxic shock syndrome, diphtheria, cholera,typhus, meningitis, whooping cough, botulism, tetanus, pyogenicinfections, dysentery, gastroenteritis, anthrax, Lyme disease, syphilis,rubella, septicemia and plague.

Effective treatment of infection may be examined in several differentways. The patient may exhibit reduced fever, reduced number oforganisms, lower level of inflammatory molecules (e.g., IFN-γ, IL-12,IL-1, TNF), and the like.

Peptide analogues of the present invention are preferably administeredas a pharmaceutical composition. Briefly, pharmaceutical compositions ofthe present invention may comprise one or more of the peptide analoguesdescribed herein, in combination with one or more physiologicallyacceptable carriers, diluents, or excipients. As noted herein, theformulation buffer used may affect the efficacy or activity of thepeptide analogue. A suitable formulation buffer contains buffer andsolubilizer. The formulation buffer may comprise buffers such as sodiumacetate, sodium citrate, neutral buffered saline, phosphate-bufferedsaline, and the like or salts, such as NaCl. Sodium acetate ispreferred. In general, an acetate buffer from 5 to 500 mM is used, andpreferably from 100 to 200 mM. The pH of the final formulation may rangefrom 3 to 10, and is preferably approximately neutral (about pH 7-8).Solubilizers, such as polyoxyethylenesorbitans (e.g., Tween 80, Tween20) and polyoxyethylene ethers (e.g., Brij 56) may also be added if thecompound is not already APS-modified.

Although the formulation buffer is exemplified herein with peptideanalogues of the present invention, this buffer is generally useful anddesirable for delivery of other peptides. Peptides that may be deliveredin this formulation buffer include indolicidin, other indolicidinanalogues (see, PCT WO 95/22338), bacteriocins, gramicidin, bactenecin,drosocin, polyphemusins, defensins, cecropins, melittins,cecropin/melittin hybrids, magainins, sapecins, apidaecins, protegrins,tachyplesins, thionins; IL-1 through 15; corticotropin-releasinghormone; human growth hormone; insulin; erythropoietin; thrombopoietin;myelin basic protein peptides; various colony stimulating factors suchas M-CSF, GM-CSF, kit ligand; and peptides and analogues of these andsimilar proteins.

Additional compounds may be included in the compositions. These include,for example, carbohydrates such as glucose, mannose, sucrose ordextrose, mannitol, other proteins, polypeptides or amino acids,chelating agents such as EDTA or glutathione, adjuvants andpreservatives. As noted herein, pharmaceutical compositions of thepresent invention may also contain one or more additional activeingredients, such as an antibiotic (see discussion herein on synergy) orcytokine.

The compositions may be administered in a delivery vehicle. For example,the composition can be encapsulated in a liposome (see, e.g., WO96/10585; WO 95/35094), complexed with lipids, encapsulated inslow-release or sustained release vehicles, such as poly-galactide, andthe like. Within other embodiments, compositions may be prepared as alyophilizate, utilizing appropriate excipients to provide stability.

Pharmaceutical compositions of the present invention may be administeredin various manners. For example, peptide analogues may be administeredby intravenous injection, intraperitoneal injection or implantation,subcutaneous injection or implantation, intradermal injection, lavage,inhalation, implantation, intramuscular injection or implantation,intrathecal injection, bladder wash-out, suppositories, pessaries,topical (e.g., creams, ointments, skin patches, eye drops, ear drops,shampoos) application, enteric, oral, or nasal route. The analogue maybe applied locally as an injection, drops, spray, tablets, cream,ointment, gel, and the like. Analogue may be administered as a bolus oras multiple doses over a period of time.

The level of peptide in serum and other tissues after administration canbe monitored by various well-established techniques such as bacterial,chromatographic or antibody based, such as ELISA, assays.

Pharmaceutical compositions of the present invention are administered ina manner appropriate to the infection or disease to be treated. Theamount and frequency of administration will be determined by factorssuch as the condition of the patient, the cause of the infection, andthe severity of the infection. Appropriate dosages may be determined byclinical trials, but will generally range from about 0.1 to 50 mg/kg.

In addition, the analogues of the present invention may be used in themanner of common disinfectants or in any situation in whichmicroorganisms are undesirable. For example, these peptides may be usedas surface disinfectants, coatings, including covalent bonding, formedical devices, coatings for clothing, such as to inhibit growth ofbacteria or repel mosquitoes, in filters for air purification, such ason an airplane, in water purification, constituents of shampoos andsoaps, food preservatives, cosmetic preservatives, media preservatives,herbicide or insecticides, constituents of building materials, such asin silicone sealant, and in animal product processing, such as curing ofanimal hides. As used herein, “medical device” refers to any device foruse in a patient, such as an implant or prosthesis. Such devicesinclude, stents, tubing, probes, cannulas, catheters, synthetic vasculargrafts, blood monitoring devices, artificial heart valves, needles, andthe like.

For these purposes, typically the peptides alone or in conjunction withan antibiotic are included in compositions commonly employed or in asuitable applicator, such as for applying to clothing. They may beincorporated or impregnated into the material during manufacture, suchas for an air filter, or otherwise applied to devices. The peptides andantibiotics need only be suspended in a solution appropriate for thedevice or article. Polymers are one type of carrier that can be used.

The analogues, especially the labeled analogues, may be used in imageanalysis and diagnostic assays or for targeting sites in eukaryoticmulticellular and single cell cellular organisms and in prokaryotes. Asa targeting system, the analogues may be coupled with other peptides,proteins, nucleic acids, antibodies and the like.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Synthesis Purification and Characterization ofPeptide Analogues

Peptide synthesis is based on the standard solid-phase Fmoc protectionstrategy. The instrument employed is a 9050 Plus PepSynthesiser(PerSeptive BioSystems Inc.). Polyethylene glycol polystyrene (PEG-PS)graft resins are employed as the solid phase, derivatized with anFmoc-protected amino acid linker for C-terminal amide synthesis. HATU(O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) is used as the coupling reagent. During synthesis,coupling steps are continuously monitored to ensure that each amino acidis incorporated in high yield. The peptide is cleaved from thesolid-phase resin using trifluoroacetic acid and appropriate scavengersand the crude peptide is purified using preparative reversed-phasechromatography.

All peptides are analyzed by mass spectrometry to ensure that theproduct has the expected molecular mass. The product should have asingle peak accounting for >95% of the total peak area when subjected toanalytical reversed-phase high performance liquid chromatography(RP-HPLC). In addition, the peptide should show a single band accountingfor >90% of the total band intensity when subjected to acid-urea gelelectrophoresis.

Peptide content, the amount of the product that is peptide rather thanretained water, salt or solvent, is measured by quantitative amino acidanalysis, free amine derivatization or spectrophotometric quantitation.Amino acid analysis also provides information on the ratio of aminoacids present in the peptide, which assists in confirming theauthenticity of the peptide.

Peptide analogues and their names are listed in Table 2. In this table,and elsewhere, the amino acids are denoted by the one-letter amino acidcode and lower case letters represent the D-form of the amino acid.

TABLE 2 10 (SEQ ID NO: 29) I L P W K W P W W P W R R 10CN (SEQ ID NO:29) I L P W K W P W W P W R R 11 (SEQ ID NO: 30) I L K K W P W W P W R RK 11CN (SEQ ID NO: 30) I L K K W P W W P W R R K 11CNR (SEQ ID NO: 31) KR R W P W W P W K K L I 11A1CN (SEQ ID NO: 22) I L K K F P F F P F R R K11A2CN (SEQ ID NO: 32) I L K K I P I I P I R R K 11A3CN (SEQ ID NO: 23)I L K K Y P Y Y P Y R R K 11A4CN (SEQ ID NO: 33) I L K K W P W P W R R K11A5CN (SEQ ID NO: 34) I L K K Y P W Y P W R R K 11A6CN (SEQ ID NO: 35)I L K K F P W F P W R R K 11A7CN (SEQ ID NO: 36) I L K K F P F W P W R RK 11ABCN (SEQ ID NO: 37) I L R Y V Y Y V Y R R K 11B1CN (SEQ ID NO: 38)I L R R W P W W P W R R K 11B2CN (SEQ ID NO: 39) I L R R W P W W P W R K11B3CN (SEQ ID NO: 40) I L K W P W W P W R R K 11B4CN (SEQ ID NO: 24) IL K K W P W W P W R K 11B5CN (SEQ ID NO: 41) I L K W P W W P W R K11B7CN (SEQ ID NO: 42) I L R W P W W P W R R K 11B7CNR (SEQ ID NO: 43) KR R W P W W P W R L I 11B8CN (SEQ ID NO: 44) I L W P W W P W R R K11B9CN (SEQ ID NO: 25) I L R R W P W W P W R R R 11B10CN (SEQ ID NO: 45)I L K K W P W W P W K K K 11B16CN (SEQ ID NO: 46) I L R W P W W P W R RK I M I L K K A G S 11B17CN (SEQ ID NO: 47) I L R W P W W P W R R K M IL K K A G S 11B18CN (SEQ ID NO: 48) I L R W P W W P W R R K D M I L K KA G S 11C3CN (SEQ ID NO: 49) I L K K W A W W P W R R K 11C4CN (SEQ IDNO: 50) I L K K W P W W A W R R K 11C5CN (SEQ ID NO: 51) W W K K W P W WP W R R K 11D1CN (SEQ ID NO: 52) L K K W P W W P W R R K 11D3CN (SEQ IDNO: 53) P W W P W R R K 11D4CN (SEQ ID NO: 54) I L K K W P W W P W R R KM I L K K A G S 11D5CN (SEQ ID NO: 55) I L K K W P W W P W R R M I L K KA G S 11D6CN (SEQ ID NO: 56) I L K K W P W W P W R R I M I L K K A G S11D11H (SEQ ID NO: 57) I L K K W P W W P W R R K M 11D12H (SEQ ID NO:58) I L K K W P W W P W R R M 11D13H (SEQ ID NO: 59) I L K K W P W W P WR R I M 11D14CN (SEQ ID NO: 60) I L K K W W W P W R K 11D15CN (SEQ IDNO: 61) I L K K W P W W W R K 11D18CN (SEQ ID NO: 26) W R I W K P K W RL P K W 11E1CN (SEQ ID NO: 62) I L K K W P W W P W R R K 11E2CN (SEQ IDNO: 63) I L K K W P W W P W R R k 11E3CN (SEQ ID NO: 64) I L K K W P W WP W R R k 11F1CN (SEQ ID NO: 65) I L K K W V W W V W R R K 11F2CN (SEQID NO: 66) I L K K W P W W V W R R K 11F3CN (SEQ ID NO: 67) I L K K W VW W P W R R K 11F4CN (SEQ ID NO: 27) I L R W V W W V W R R K 11F4CNR(SEQ ID NO: 68) K R R W V W W V W R L I 11G2CN (SEQ ID NO: 69) I K K W PW W P W R R K 11G3CN (SEQ ID NO: 70) I L K K P W W P W R R K 11G4CN (SEQID NO: 71) I L K K W W W P W R R K 11G5CN (SEQ ID NO: 72) I L K K W P WW W R R K 11G6CN (SEQ ID NO: 73) I L K K W P W W P R R K 11G7CN (SEQ IDNO: 74) I L K K W P W W P W R R 11G13CN (SEQ ID NO: 28) I L K K W P W WP W K 11G14CN (SEQ ID NO: 75) I L K K W P W W P W R 11H1CN (SEQ ID NO:76) A L R W P W W P W R P K 11H2CN (SEQ ID NO: 77) I A R W P W W P W R RK 11H3CN (SEQ ID NO: 78) I L A W P W W P W R R K 11H4CN (SEQ ID NO: 79)I L R A P W W P W R R K 11H5CN (SEQ ID NO: 80) I L R W A W W P W R R K11H6CN (SEQ ID NO: 81) I L R W P A W P W R R K 11H7CN (SEQ ID NO: 82) IL R W P W A P W R R K 11H8CN (SEQ ID NO: 83) I L R W P W W A W R R K11H9CN (SEQ ID NO: 84) I L R W P W W P A R R K 11H10CN (SEQ ID NO: 85) IL R W P W W P W A R K 11H11CN (SEQ ID NO: 86) I L R W P W W P W R A K11H12CN (SEQ ID NO: 87) I L R W P W W P W R R A CN suffix = amidatedC-terminus H suffix = humoserine at C-terminus R suffix= retro-synthesized peptide

Example 2 Synthesis of Modified Peptides

Indolicidin analogues are modified to alter the physical properties ofthe original peptide. Such modifications include: acetylation at theN-terminus, Fmoc-derivatized N-terminus, polymethylation,peracetylation, and branched derivatives.

α-N-terminal acetylation. Prior to cleaving the peptide from the resinand deptrotecting it, the fully protected peptide is treated withN-acetylimidazole in DMF for 1 hour at room temperature, which resultsin selective reaction at the α-N-terminus. The peptide is thendeprotected/cleaved and purified as for an unmodified peptide.

Fmoc-derivatized α-N-terminus. If the final Fmoc deprotection step isnot carried, the α-N-terminus Fmoc group remains on the peptide. Thepeptide is then side-chain deprotected/cleaved and purified as for anunmodified peptide.

Polymethylation. The purified peptide in a methanol solution is treatedwith excess sodium bicarbonate, followed by excess methyl iodide. Thereaction mixture is stirred overnight at room temperature, extractedwith organic solvent, neutralized and purified as for an unmodifiedpeptide. Using this procedure, a peptide is not fully methylated;methylation of MBI 11CN yielded an average of 6 methyl groups. Thus, themodified peptide is a mixture of methylated products.

Peracetylation. A purified peptide in DMF solution is treated withN-acetylimidazole for 1 hour at room temperature. The crude product isconcentrated, dissolved in water, lyophilized, re-dissolved in water andpurified as for an unmodified peptide. Complete acetylation of primaryamine groups is observed.

Four/eight branch derivatives. The branched peptides are synthesized ona four or eight branched core bound to the resin. Synthesis anddeprotection/cleavage proceed as for an unmodified peptide. Thesepeptides are purified by dialysis against 4 M guanidine hydrochloridethen water, and analyzed by mass spectrometry.

Peptides modified using the above procedures are listed in Table 3.

TABLE 3 Peptide Peptide modified name Sequence Modification 10 10A I L PW K W P W W P W R R Acetylated α-N-terminus (SEQ ID NO: 29) 11 11 I L KK W P W W P W R R K Acetylated α-N-terminus (SEQ ID NO: 30) 11CN 11CAN IL K K W P W W P W R R K Acetylated α-N-terminus (SEQ ID NO: 30) 11CN11CNW1 I L K K W P W W P W R R K Fmoc-derivatized N-terminus (SEQ ID NO:30) 11CN 11CNX1 I L K K W P W W P W R R K Polymethylated derivative (SEQID NO: 30) 11CN 11CNY1 I L K K W P W W P W R P K Peracetylatedderivative (SEQ ID NO: 30) 11 11M4 I L K K W P W W P W R R K Four branchderivative (SEQ ID NO: 30) 11 11M8 I L K K W P W W P W R R K Eightbranch derivative (SEQ ID NO: 30) 11B1CN 11B1CNW1 I L R R W P W W P W RR K Fmoc-derivatized N-terminus (SEQ ID NO: 38) 11B4CN 11B4ACN I L K K WP W W P W R K Acetylated N-terminus (SEQ ID NO: 24) 11B9CN 11B9ACN I L RR W P W W P W R R R Acetylated N-terminus (SEQ ID NO: 25) 11D9 11D9M8 WW P W R R K Eight branch derivative (SEQ ID NO: 88) 11D10 11D10M8 I L KK W P W Eight branch derivative (SEQ ID NO: 89) 11G6CN 11G6ACN I L K K WP W W P R P K Acetylated α-N-terminus (SEQ ID NO: 73) 11G7CN 11G7ACN I LK K W P W W P W R R Acetylated α-N-terminus (SEQ ID NO: 74)

Example 3 Recombinant Production of Peptide Analogues

Peptide analogues are alternatively produced by recombinant DNAtechnique in bacterial host cells. The peptide is produced as a fusionprotein, chosen to assist in transporting the fusion peptide toinclusion bodies, periplasm, outer membrane or extracellularenvironment.

Construction of Plasmids Encoding MBI-11 Peptide Fusion Protein

Amplification by polymerase chain reaction is used to synthesizedouble-stranded DNA encoding the MBI peptide genes from single-strandedtemplates. For MBI-11, 100 μl of reaction mix is prepared containing 50to 100 ng of template, 25 pmole of each primer, 1.5 mM MgCl₂, 200 μM ofeach dNTP, 2U of Taq polymerase in the supplier's buffer. The reactionsproceeded with 25 cycles of 94° C. for 30 sec., 55° C. for 30 sec., 74°C. for 30 sec., followed by 74° C. for 1 min. Amplified product isdigested with BamHI and HindIII and cloned into a plasmid expressionvector encoding the fusion partner and a suitable selection marker.

Production of MBI-11 Peptide Fusion in E. coli

The plasmid pR2h-11, employing a T7 promoter, high copy origin ofreplication, Ap^(r) marker and containing the gene of the fusionprotein, is co-electroporated with pGP1-2 into E. coli strain XL1-Blue.Plasmid pGP1-2 contains a T7 RNA polymerase gene under control of alambda promoter and cI857 repressor gene. Fusion protein expression isinduced by a temperature shift from 30° C. to 42° C. Inclusion bodiesare washed with solution containing solubilizer and extracted withorganic extraction solvent. Profiles of the samples are analyzed bySDS-PAGE. FIG. 1 shows the SDS-PAGE analysis and an extraction profileof inclusion body from whole cell. The major contaminant in the organicsolvent extracted material is β-lactamase (FIG. 1). The expression levelin these cells is presented in Table 4.

TABLE 4 % which is Fusion Mol.mass % protein in % in inclusion MBI-11protein (kDa) whole cell lysate body extract peptide MBI-11 20.1 15 427.2

In addition, a low-copy-number vector, pPD100, which contains achloramphenicol resistance gene, is used to express MBI-11 in order toeliminate the need for using ampicillin, thereby reducing the appearanceof β-lactamase in extracted material. This plasmid allows selective geneexpression and high-level protein overproduction in E. coli using thebacteriophage T7 RNA polymerase/T7 promoter system (Dersch et al., FEMSMicrobiol. Lett. 123: 19-26,1994). pPD100 contains a chloramphenicolresistance gene (CAT) as a selective marker, a multiple cloning site,and an ori sequence derived from the low-copy-number vector pSC101.There are only about 4 to 6 copies of these plasmids per host cell. Theresulting construct containing MBI-11 is called pPDR2h-11. FIG. 2presents a gel electrophoresis analysis of the MBI-11 fusion proteinexpressed in this vector. Expression level of MBI-11 fusion protein iscomparable with that obtained from plasmid pR2h-11. The CAT gene productis not apparent, presumably due to the low-copy-number nature of thisplasmid, CAT protein is not expressed at high levels in pPDR2h-11.

Example 4 In vitro Assays to Measure Peptide Analogue Activity

Agarose Dilution Assay

The agarose dilution assay measures antimicrobial activity of peptidesand peptide analogues, which is expressed as the minimum inhibitoryconcentration (MIC) of the peptides.

In order to mimic in vivo conditions, calcium and magnesium supplementedMueller Hinton broth is used in combination with a low EEO agarose asthe bacterial growth medium. The more commonly used agar is replacedwith agarose as the charged groups in agar prevent peptide diffusionthrough the media. The media is autoclaved and then cooled to 50-55° C.in a water bath before aseptic addition of antimicrobial solutions. Thesame volume of different concentrations of peptide solution are added tothe cooled molten agarose that is then poured to a depth of 3-4 mm.

The bacterial inoculum is adjusted to a 0.5 McFarland turbidity standard(PML Microbiological) and then diluted 1:10 before application on to theagarose plate. The final inoculum applied to the agarose isapproximately 10⁴ CFU in a 5-8 mm diameter spot. The agarose plates areincubated at 35-37° C. for 16 to 20 hours.

The MIC is recorded as the lowest concentration of peptide thatcompletely inhibits growth of the organism as determined by visualinspection. Representative MICs for various indolicidin analogues areshown in the Table 5 below.

TABLE 5 Organism Organism # MIC (μg/ml) 1. MBI 10 A. calcoaceticus AC001128 E. coli ECO002 128 E. faecalis EFS004 8 K. pneumoniae KP001 128 P.aeruginosa PA003 >128 S. aureus SA007 2 S. maltophilia SMA001 128 S.marcescens SMS003 >128 2. MBI 10A E. faecalis EFS004 16 E. faeciumEFM003 8 S. aureus SA010 8 3. MBI 10CN A. calcoaceticus AC001 64 E.cloacae ECL007 >128 E. coli ECO001 32 E. coli SBECO2 16 E. faecalisEFS004 8 E. faecium EFM003 2 K. pneumoniae KP002 64 P. aeruginosaPA002 >128 S. aureus SA003 2 S. epiderimidis SE010 4 S. maltophiliaSMA002 64 S. marcescens SMS004 >128 4. MBI 11 A. calcoaceticus AC002 8E. cloacae ECL007 >128 E. coli ECO002 64 E. faecium EFM003 4 E. faecalisEFS002 64 K. pneumoniae KP001 128 P. aeruginosa PA004 >128 S. aureusSA004 4 S. maltophilia SMA002 128 S. marcescens SMS004 >128 5. MBI 11AA. calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coli ECO005 >64 E.faecalis EFS004 32 K. pneumoniae KP001 64 P. aeruginosa PA024 >64 S.aureus SA002 4 S. maltophilia SMA002 >64 S. marcescens SMS003 >64 6. MBI11ACN A. calcoaceticus AC002 2 E. cloacae ECL007 >128 E. coli ECO005 16E. faecalis EFS004 8 E. faecalis EFS008 64 K. pneumoniae KP001 16 P.aeruginosa PA004 >128 S. aureus SA014 8 S. epidermidis SE010 4 S.maltophilia SMA002 64 S. marcescens SMS003 >128 7. MBI 11CN A.calcoaceticus AC001 128 E. cloacae ECL007 >64 E. coli ECO002 8 E.faecium EFM001 8 E. faecalis EFS001 32 H. influenzae HIN001 >128 K.pneumoniae KP002 128 P. aeruginosa PA003 >128 P. mirabilis PM002 >128 S.aureus SA003 2 S. marcescens SBSM1 >128 S. pneumoniae SBSPN2 >128 S.epidermidis SE001 2 S. maltophilia SMA001 64 S. marcescens SMS003 >128S. pyogenes SPY003 8 8. MBI 11CNR A. calcoaceticus AC002 4 E. cloacaeECL007 >128 E. coli ECO005 8 E. faecalis EFS001 4 K. pneumoniae KP001 4P. aeruginosa PA004 32 S. aureus SA093 4 S. epidermidis SE010 4 S.maltophilia SMA002 32 S. marcescens SMS003 128 9. MBI 11CNW1 A.calcoaceticus AC002 8 E. cloacae ECL007 64 E. coli ECO005 32 E. faecalisEFS001 8 K. pneumoniae KP001 32 P. aeruginosa PA004 64 S. aureus SA010 4S. maltophilia SMA002 32 S. marcescens SMS003 >128 10. MBI 11CNX1 A.calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coli ECO005 64 E.faecalis EFS004 16 K. pneumoniae KP001 >64 P. aeruginosa PA024 >64 S.aureus SA006 2 S. maltophilia SMA002 >64 S. marcescens SMS003 >64 11.MBI 11CNY1 A. calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coliECO005 >64 E. faecalis EFS004 >64 K. pneumoniae KP001 >64 P. aeruginosaPA004 >64 S. aureus SA006 16 S. epidermidis SE010 128 S. maltophiliaSMA002 >64 S. marcescens SMS003 >64 12. MBI 11M4 E. faecium EFM001 32 E.faecalis EFS001 32 S. aureus SA008 8 13. MBI 11M8 E. faecalis EFS002 32E. faecium EFM002 32 S. aureus SA008 32 14. MBI 11A1CN A. calcoaceticusAC002 16 E. cloacae ECL007 >128 E. coli ECO002 32 E. faecium EFM002 1 E.faecalis EFS002 32 H. influenzae HIN002 >128 K. pneumoniae KP002 >128 P.aeruginosa PA004 >128 S. aureus SA005 8 P. vulgaris SBPV1 >128 S.marcescens SBSM2 >128 S. pneumoniae SBSPN2 >128 S. epidermidis SE002 16S. maltophilia SMA002 >128 15. MBI 11A2CN A. calcoaceticus AC001 >128 E.cloacae ECL007 >128 E. coli ECO003 >128 E. faecium EFM003 16 E. faecalisEFS002 >128 K. pneumoniae KP002 >128 P. aeruginosa PA004 >128 S. aureusSA004 8 S. maltophilia SMA001 >128 S. marcescens SMS003 >128 16. MBI11A3CN A. calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coliECO002 >128 E. faecium EFM003 64 E. faecalis EFS002 >128 H. influenzaeHIN002 >128 K. pneumoniae KP001 >128 P. aeruginosa PA002 >128 S. aureusSA004 32 P. vulgaris SBPV1 >128 S. marcescens SBSM2 >128 S. pneumoniaeSBSPN3 >128 S. epidermidis SE002 128 S. maltophilia SMA001 >128 17. MBI11A4CN A. calcoaceticus AC002 8 E. cloacae ECL007 >128 E. coli ECO003 32E. faecalis EFS002 64 E. faecium EFM001 32 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA005 2 S. epidermidis SE002 8 S.maltophilia SMA002 >128 S. marcescens SMS004 >128 18. MBI 11A5CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO003 128 E.faecium EFM003 4 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA003 >128 S. aureus SA002 16 S. maltophilia SMA002 >128 S.marcescens SMS003 >128 19. MBI 11A6CN E. faecium EFM003 2 E. faecalisEFS004 64 S. aureus SA016 2 20. MBI 11A7CN E. faecium EFM003 2 E.faecalis EFS002 16 S. aureus SA009 2 21. MBI 11A8CN A. calcoaceticusAC002 8 E. cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS001 4 K.pneumoniae KP001 128 P. aeruginosa PA004 >128 S. aureus SA093 1 S.epidermidis SE010 16 S. maltophilia SMA002 32 S. marcescens SMS003 >12822. MBI 11B1CN A. calcoaceticus AC001 32 E. cloacae ECL007 >128 E. coliECO003 8 E. faecium EFM002 2 E. faecalis EFS004 8 K. pneumoniae KP002 64P. aeruginosa PA005 >128 S. aureus SA005 2 S. epidermidis SE001 2 S.maltophilia SMA001 64 S. marcescens SMS004 >128 23. MBI 11B1CNW1 A.calcoaceticus AC002 16 E. cloacae ECL007 64 E. coli ECO005 32 E.faecalis EFS004 8 K. pneumoniae KP001 32 P. aeruginosa PA004 64 S.aureus SA014 16 S. epidermidis SE010 8 S. maltophilia SMA002 32 S.marcescens SMS003 >128 24. MBI 11B2CN A. calcoaceticus AC001 64 E.cloacae ECL007 >128 E. coli ECO003 16 E. faecium EFM001 8 E. faecalisEFS004 8 K. pneumoniae KP002 64 P. aeruginosa PA003 >128 S. aureus SA0052 S. maltophilia SMA002 64 S. marcescens SMS004 >128 25. MBI 11B3CN A.calcoaceticus AC001 64 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 8 E. faecalis EFS001 16 K. pneumoniae KP002 64 P.aeruginosa PA003 >128 S. aureus SA010 4 S. maltophilia SMA002 32 S.marcescens SMS004 >128 26. MBI 11B4CN A. calcoaceticus AC001 >128 E.cloacae ECL007 >128 E. coli ECO003 16 E. faecalis EFS002 16 H.influenzae HIN002 >128 K. pneumoniae KP002 128 P. aeruginosa PA006 >128S. aureus SA004 2 S. marcescens SBSM2 >128 S. pneumoniae SBSPN3 128 S.epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescens SMS004 >12827. MBI 11B4ACN A. calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coliECO005 32 E. faecalis EFS008 64 K. pneumoniae KP001 32 P. aeruginosaPA004 >128 S. aureus SA008 1 S. epiderimidis SE010 8 S. maltophiliaSMA002 64 S. marcescens SMS003 >128 28. MBI 11B5CN E. faecium EFM002 1E. faecalis EFS002 16 S. aureus SA005 2 29. MBI 11B7 A. calcoaceticusAC002 4 E. cloacae ECL007 >128 E. coli ECO005 16 E. faecalis EFS008 8 K.pneumoniae KP001 16 P. aeruginosa PA004 >128 S. aureus SA093 1 S.epidermidis SE010 4 S. maltophilia SMA002 64 S. marcescens SMS003 >12830. MBI 11B7CN A. calcoaceticus AC003 32 E. cloacae ECL009 32 E. coliECO002 8 E. faecium EFM001 4 E. faecalis EFS004 4 H. influenzaeHIN002 >128 K. pneumoniae KP001 32 P. aeruginosa PA004 128 P. mirabilisPM002 >128 S. aureus SA009 2 S. marcescens SBSM1 >128 S. pneumoniaeSBSPN3 >128 S. epidermidis SE003 2 S. maltophilia SMA004 128 S. pyogenesSPY006 16 31. MBI 11B7CNR A. calcoaceticus AC002 4 E. cloacae ECL007 64E. coli ECO005 8 E. faecalis EFS001 4 K. pneumoniae KP001 8 P.aeruginosa PA004 64 S. aureus SA093 2 S. epidermidis SE010 4 S.maltophilia SMA002 32 S. marcescens SMS003 >128 32. MBI 11B8CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 16 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA005 >128 S. aureus SA009 4 S. epidermidis SF002 4 S.maltophilia SMA002 128 S. marcescens SMS003 >128 33. MBI 11B9CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 8 E. faeciumEFM002 4 E. faecalis EFS002 8 H. influenzae HIN002 >128 K. pneumoniaeKP001 32 P. aeruginosa PA004 128 P. mirabilis PM002 >128 S. aureus SA0104 S. pneumoniae SBSPN2 >128 S. epidermidis SE010 2 S. maltophilia SMA00232 S. marcescens SMS003 >128 S. pneumoniae SPN044 >128 S. pyogenesSPY005 16 34. MBI 11B9ACN A. calcoaceticus AC001 32 E. cloacaeECL007 >128 E. coli ECO003 8 E. faecium EFM001 4 E. faecalis EFS004 8 K.pneumoniae KP002 32 P. aeruginosa PA005 >128 S. aureus SA019 2 S.epidermidis SE002 2 S. maltophilia SMA001 16 S. marcescens SMS004 >12835. MBI 11B10CN E. faecium EFM003 4 E. faecalis EFS002 64 S. aureusSA008 2 36. MBI 11B16CN A. calcoaceticus AC002 4 E. cloacae ECL007 >128E. coli ECO005 16 E. faecalis EFS001 2 K. pneumoniae KP001 16 P.aeruginosa PA004 >128 S. aureus SA093 2 S. epidemidis SE010 4 S.maltophilia SMA002 32 S. marscescens SMS003 >128 37. MBI 11B17CN A.calcoaceticus AC002 2 E. cloacae ECL007 >128 E. coli ECO005 8 E.faecalis EFS008 4 K. pneumoniae KP001 16 P. aeruginosa PA004 >128 S.aureus SA093 2 S. epidermidis SE010 4 S. maltophilia SMA002 32 S.marcescens SMS003 >128 38. MBI 11B18CN A. calcoaceticus AC002 2 E.cloacae ECL007 >128 E. coli ECO005 32 E. faecalis EFS008 4 K. pneunoniaeKP001 32 P. aeruginosa PA004 >128 S. aureus SA093 2 S. epidermidis SE0104 S. maltophilia SMA002 64 S. marcescens SMS003 >128 39. MBI 11C3CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM002 1 E. faecalis EFS002 32 K. pneumoniae KP001 128 P.aeruginosa PA005 >128 S. aureus SA005 2 S. epidermidis SE002 2 S.maltophilia SMA002 64 S. marcescens SMS004 >128 40. MBI 11C4CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 32 E.faecium EFM003 2 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA005 >128 S. aureus SA009 4 S. epidermidis SE002 4 S.maltophilia SMA002 64 S. marcescens SMS004 >128 41. MBI 11C5CN A.calcoaceticus AC001 32 E. cloacae ECL007 >128 E. coli ECO001 8 E.faecium EFM003 2 E. faecalis EFS002 16 K. pneumoniae KP002 16 P.aeruginosa PA003 64 S. aureus SA009 2 S. epidermidis SE002 2 S.maltophilia SMA002 16 S. marcescens SMS004 >128 42. MBI 11D1CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 16 E.faecium EFM001 16 E. faecalis EFS002 32 K. pneumoniae KP002 64 P.aeruginosa PA003 >128 S. aureus SA004 2 S. epidermidis SE010 8 S.maltophilia SMA001 64 S. marcescens SMS003 >128 43. MBI 11D3CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 64 E.faecium EFM003 8 E. faecalis EFS002 32 K. pneumoniae KP002 >128 P.aeruginosa PA024 >128 S. aureus SA009 8 S. maltophilia SMA001 64 S.marcescens SMS004 >128 44. MBI 11D4CN A. calcoaceticus AC001 >64 E.cloacae ECL007 >64 E. coli ECO003 64 E. faecium EFM002 1 E. faecalisEFS002 16 K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S. aureusSA009 4 S. maltophilia SMA001 >64 S. marcescens SMS004 >64 45. MBI11D5CN A. calcoaceticus AC001 >64 E. cloacae ECL007 >64 E. coli ECO00364 E. faecium EFM003 1 E. faecalis EFS002 16 K. pneunoniae KP001 >64 P.aeruginosa PA003 >64 S. aureus SA005 8 S. maltophilia SMA001 64 S.marcescens SMS004 >64 46. MBI 11D6CN A. calcoaceticus AC002 4 E. cloacaeECL007 >32 E. coli ECO002 32 E. faecium EFM003 1 E. faecalis EFS002 4 K.pneumoniae KP002 >64 P. aeruginosa PA024 >64 S. aureus SA009 8 S.epidermidis SE010 4 S. maltophpilia SMA001 >64 S. marcescens SMS004 >6447. MBI 11D9M8 E. faecium EFM002 32 S. aureus SA007 32 E. faecalisEFS002 128 S. aureus SA016 128 48. MBI 11D10M8 E. faecium EFM003 32 E.faecalis EFS002 32 S. aureus SA008 32 49. MBI 11D11H A. calcoaceticusAC001 >64 E. cloacae ECL007 >64 E. coli ECO002 32 K. pneumoniaeKP001 >64 P. aeruginosa PA001 >64 S. aureus SA008 4 S. maltophiliaSMA002 >64 S. marcescens SMS004 >64 50. MBI 11D12H A. calcoaceticusAC001 >64 E. cloacae ECL007 >64 E. coli ECO003 64 E. faecalis EFS004 16K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S. aureus SA014 16 S.maltophilia SMA002 >64 S. marcescens SMS004 >64 51. MBI 11D13H A.calcoaceticus AC001 64 E. cloacae ECL007 >64 E. coli ECO002 32 E.faecalis EFS004 16 K. pneumoniae KP002 >64 P. aeruginosa PA004 >64 S.aureus SA025 4 S. maltophilia SMA002 >64 S. marcescens SMS004 >64 52.MBI 11D14CN E. faecium EFM003 1 E. faecalis EFS002 32 S. aureus SA009 453. MBI 11D15CN E. faecium EFM003 4 E. faecalis EFS002 32 S. aureusSA009 8 54. MBI 11D18CN A. calcoaceticus AC003 32 E. cloacae ECL009 64E. coli ECO002 4 E. faecium EFM003 2 E. faecalis EFS002 32 H. influenzaeHIN002 >128 K. pneumoniae KP002 64 P. aeruginosa PA006 >128 P. mirabilisPM003 >128 S. aureus SA010 4 P. vulgaris SBPV1 32 S. marcescensSBSM2 >128 S. pneumoniae SBSPN3 64 S. epidermidis SE010 2 S. maltophiliaSMA003 16 S. pyogenes SPY003 32 55. MBI 11E1CN A. calcoaceticus AC001 32E. cloacae ECL007 >128 E. coli ECO003 8 E. faecium EFM001 8 E. faecalisEFS002 8 K. pneumoniae KP002 32 P. aeruginosa PA003 128 S. aureus SA0061 S. maltophilia SMA001 64 S. marcescens SMS003 >128 56. MBI 11E2CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 8 E. faeciumEFM001 16 E. faecalis EFS002 32 K. pneumoniae KP002 64 P. aeruginosaPA001 >128 S. aureus SA016 2 S. epidermidis SE010 4 S. maltophiliaSMA001 64 S. marcescens SMS004 >128 57. MBI 11E3CN A. calcoaceticusAC001 16 E. cloacae ECL007 >128 E. coli ECO001 4 E. faecium EFM003 2 E.faecalis EFS004 8 H. influenzae HIN002 >128 K. pneumoniae KP002 32 P.aeruginosa PA041 64 P. mirabilis PM001 >128 S. aureus SA010 2 S.pneumoniae SBSPN2 >128 S. epidermidis SE002 1 S. maltophilia SMA001 32S. marcescens SMS004 >128 S. pneumoniae SPN044 >128 S. pyogenes SPY00216 58. MBI 11F1CN E. cloacae ECL007 >128 E. coli ECO003 8 E. faeciumEFM003 2 E. faecalis EFS004 16 K. pneumoniae KP002 32 P. aeruginosaPA004 64 S. aureus SA009 2 S. marcescens SBSM1 >128 S. marcescensSMS003 >128 59. MBI 11F2CN A. calcoaceticus AC002 4 E. coli ECO002 8 E.faecium EFM002 4 E. faecalis EFS002 32 K. pneumoniae KP002 128 P.aeruginosa PA005 >128 S. aureus SA012 4 S. epidermidis SE002 4 S.maltophilia SMA002 64 S. marcescens SMS004 >128 60. MBI 11F3CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO002 8 E. faeciumEFM003 4 E. faecalis EFS002 8 H. influenzae HIN002 >128 K. pneumoniaeKP002 64 P. aeruginosa PA041 128 S. aureus SA005 2 S. pneumoniaeSBSPN3 >128 S. epidermidis SE003 2 S. maltophilia SMA002 64 S.marcescens SMS004 >128 S. pneumoniae SPN044 >128 S. pyogenes SPY006 861. MBI 11F4CN A. calcoaceticus AC003 16 E cloacae ECL006 16 E. coliECO001 8 E. faecalis EFS004 8 H. influenzae HIN003 >128 K. pneumoniaeKP001 8 P. aeruginosa PA020 32 S. aureus SA007 I S. marcescensSBSM1 >128 S. pneumoniae SBSPN3 >128 S. epidermidis SE010 2 S.maltophilia SMA006 16 S. pyogenes SPY005 32 62. MBI 11F4CNR A.calcoaceticus AC002 16 E. cloacae ECL007 32 E. coli ECO005 32 E.faecalis EFS008 32 K. pneumoniae KP001 32 P. aeruginosa PA004 64 S.aureus SA093 8 S. epidermidis SE010 8 S. maltophilia SMA002 32 S.marcescens SMS003 >128 63. MBI 11G2CN E. cloacae ECL007 >128 E. coliECO003 16 E. faecium EFM002 4 E. faecalis EFS004 16 K. pneumoniae KP002128 P. aeruginosa PA004 >128 S. aureus SA009 2 S. maltophiliaSMA001 >128 S. marcescens SMS004 >128 64. MBI 11G3CN E. cloacaeECL007 >128 E. coli ECO003 64 E. faecium EFM002 32 E. faecalis EFS002 64K. pneumoniae KP001 >128 P. aeruginosa PA003 >128 S. aureus SA009 8 S.maltophilia SMA001 >128 S. marcescens SMS004 >128 65. MBI 11G4CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 32 E.faecium EFM003 1 E. faecalis EFS002 32 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA004 1 S. epidermidis SE010 2 S.maltophilia SMA002 64 S. marcescens SMS003 >128 66. MBI 11G5CN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO003 16 E.faecium EFM002 8 E. faecalis EFS002 16 K. pnuemoniae KP001 >128 P.aeruginosa PA003 >128 S. aureus SA012 4 S. epidermidis SE002 2 S.maltophilia SMA002 64 S. marcescens SMS004 >128 67. MBI 11G6CN A.calcoaceticus AC001 >128 E. cloacae ECL007 >128 E. coli ECO002 32 E.faecium EFM003 4 E. faecalis EFS002 128 K. pneumoniae KP001 >128 P.aeruginosa PA004 >128 S. aureus SA006 2 S. epidermidis SE002 8 S.maltophilia SMA001 >128 S. marcescens SMS003 >128 68. MBI 11G6ACN A.calcoaceticus AC002 4 E. cloacae ECL007 >128 E. coli ECO005 64 E.faecalis EFS008 >128 K. pneumoniae KP001 >128 P. aeruginosa PA004 >128S. aureus SA014 64 S epidermidis SE010 32 S. maltophilia SMA002 >128 S.marcescens SMS003 >128 69. MBI 11G7CN A. calcoaceticus AC001 128 E.cloacae ECL006 64 E. coli ECO005 8 E. faecium EFM001 8 E. faecalisEFS002 32 H. influenzae HIN002 >128 K. pneumoniae KP001 16 P. aeruginosaPA006 >128 S. aureus SA012 2 H. influenzae SBHIN2 >128 S. marcescensSBSM1 >128 S. pneumoniae SBSPN2 >128 S. epidermidis SE002 2 S.maltophilia SMA001 32 S. marcescens SMS003 >128 S. pneumoniaeSPN044 >128 S. pyogenes SPY006 16 70. MBI 11G7ACN A. calcoaceticus AC0024 E. cloacae ECL007 >32 E. coli ECO002 16 E. faecium EFM001 8 E.faecalis EFS008 32 K. pneumoniae KP002 >32 P. aeruginosa PA006 >32 S.aureus SA010 1 S. epidermidis SE002 4 S. maltophilia SMA001 32 S.marcescens SMS004 >32 71. MBI 11G13CN E. coli ECO002 32 E. faeciumEFM002 16 E. faecalis EFS002 64 H. influenzae HIN002 >128 P. aeruginosaPA004 >128 S. aureus SA004 4 E. coli SBECO3 32 S. marcescens SBSM1 >128S. pneumoniae SBSPN3 128 72. MBI 11G14CN A. calcoaceticus AC002 8 E.cloacae ECL007 >128 E. coli ECO003 32 E. faecium EFM001 16 E. faecalisEFS002 32 K. pneumoniae KP002 128 P. aeruginosa PA006 >128 S. aureusSA013 0.5 S. epidermidis SE002 8 S. maltophilia SMA002 128 S. marcescensSMS004 >128 73. MBI 11G16CN A. calcoaceticus AC002 8 E. cloacaeECL007 >128 E. coli ECO005 16 E. faecalis EFS008 16 K. pneumoniae KP00116 P. aeruginosa PA004 128 S. aureus SA093 2 S. epidermidis SE010 4 S.maltophilia SMA002 64 S. marcescens SMS003 >128Broth Dilution Assay

This assay also uses calcium and magnesium supplemented Mueller Hintonbroth as the growth medium. Typically 100 μl of broth is dispensed intoeach well of a 96-well microtitre plate and 100 μl volumes of two-foldserial dilutions of the peptide analogue are made across the plate. Onerow of wells receives no peptide and is used as a growth control. Eachwell is inoculated with approximately 5×10⁵ CFU of bacteria and theplate is incubated at 35-37° C. for 16-20 hours. The MIC is againrecorded at the lowest concentration of peptide that completely inhibitsgrowth of the organism as determined by visual inspection.

For example, MIC values were established for a series of peptideanalogues against S. aureus strains. Results are shown in Table 6 below.

TABLE 6 MIC (μg/ml) MBI MBI MBI MBI MBI MBI MBI Organism Organism # 10CN11CN 11A1CN 11A2CN 11B1CN 11B2CN 11B7CN Gram-negative: A. calcoaceticusAC001 64 256 >256 >256 64 128 64 E. cloacae ECL007256 >256 >256 >256 >256 >256 >256 E. coli ECO005 64 128 >256 >256 64 6464 K. pneumoniae KP001 64 >256 >256 >256 >256 >256 256 P. aeruginosaPA004 >256 256 >256 >256 64 256 256 S. maltophilia SMA002 6464 >256 >256 32 32 32 S. marcescensSMS003 >256 >256 >256 >256 >256 >256 >256 Gram-positive: E. faecalisEFS004 64 128 >256 >256 64 64 64 S. aureus SA002 16 64 >256 >256 32 3216 S. epidermidis SE005 8 8 16 256 4 4 4Time Kill Assay

Time kill curves are used to determine the antimicrobial activity ofcationic peptides over a time interval. Briefly, in this assay, asuspension of microorganisms equivalent to a 0.5 McFarland Standard isprepared in 0.9% saline. This suspension is then diluted such that whenadded to a total volume of 9 ml of cation-adjusted Mueller Hinton broth,the inoculum size is 1×10⁶ CFU/ml. An aliquot of 0.1 ml is removed fromeach tube at pre-determined intervals up to 24 hours, diluted in 0.9%saline and plated in triplicate to determine viable colony counts. Thenumber of bacteria remaining in each sample is plotted over time todetermine the rate of cationic peptide killing. Generally a three ormore log₁₀ reduction in bacterial counts in the antimicrobial suspensioncompared to the growth controls indicate an adequate bactericidalresponse.

As shown in FIG. 3, all peptides demonstrated a three or more log₁₀reduction in bacterial counts in the antimicrobial suspension comparedto the growth controls indicating that these peptides have met thecriteria for a bactericidal response.

Synergy Assay

Treatment with a combination of peptide analogues and conventionalantibiotics can have a synergistic effect. Synergy is assayed using theagarose dilution technique, where an array of plates, each containing acombination of peptide and antibiotic in a unique concentration mix, isinoculated with the bacterial isolates. Synergy is investigated forpeptide analogues in combination with a number of conventionalantibiotics including, but not limited to, penicillins, cephalosporins,carbapenems, monobactams, aminoglycosides, macrolides, fluoroquinolones.

Synergy is expressed as a Fractional Inhibitory Concentration (FIC),which is calculated according to the equation below. An FIC of less thanor equal to 0.5 is evidence of synergy, although combinations withhigher values may be therapeutically useful.

${FIC} = {\frac{{MIC}\left( {{peptide}\mspace{14mu}{in}\mspace{14mu}{combination}} \right)}{{MIC}\left( {{peptide}\mspace{14mu}{alone}} \right)} + \frac{{MIC}\left( {{antibiotic}\mspace{14mu}{in}\mspace{14mu}{combination}} \right)}{{MIC}\left( {{antibiotic}\mspace{14mu}{alone}} \right)}}$

Table 7 shows exemplary synergy data for combinations of indolicidinanalogues and Mupirocin.

TABLE 7 Mupirocin Mupirocin Peptide Peptide MIC Comb. MIC MIC Comb. MICPeptide Organism (μg/ml) (μg/ml) (μg/ml) (μg/ml) FIC MBI 11A1CN E. coliECO1 >100 10 32 4 0.14 MBI 11A1CN E. faecalis EFS8 100 100 >128 >128 2MBI 11A1CN P. aeruginosa PA3 >100 >100 >128 >128 2 MBI 11A1CN S. aureusSBSA3 100 100 >128 >128 2 MBI 11A1CN S. aureus SBSA5 30 10 128 32 0.58MBI 11A1CN S. marcescens SBSM1 >100 >100 >128 >128 2 MBI 11A3CN E. coliSBECO1 100 30 64 8 0.43 MBI 11A3CN E. faecalis EFS8 100 100 >128 >128 2MBI 11A3CN P. aeruginosa PA3 >100 >100 >128 >128 2 MBI 11A3CN S. aureusSBSA2 >100 >100 128 128 2 MBI 11A3CN S. marcescensSBSM2 >100 >100 >128 >128 2 MBI 11B4CN E. coli ECO1 >100 10 16 4 0.26MBI 11B4CN E. faecalis EFS8 100 100 64 64 2 MBI 11B4CN S. aureus SBSA3100 10 32 16 0.60 MBI 11B4CN S. aureus SBSA4 >100 >100 8 8 2 MBI 11B4CNS. marcescens SBSM1 >100 >100 >128 >128 2 MBI 11D18CN E. coliSBEC02 >100 10 16 1 0.07 MBI 11D18CN E. faecalis EFS8 100 100 16 16 2MBI 11D18CN P. aeruginosa PA2 >100 30 128 64 0.53 MBI 11D18CN P.aeruginosa PA24 >100 >100 >128 >128 2 MBI 11D18CN P. vulgaris SBPV1 3 332 4 1.13 MBI 11D18CN S. aureus SBSA4 >100 0.1 16 2 0.13 MBI 11D18CN S.marcescens SBSM1 >100 30 >128 64 0.28 MBI 11G13CN E. coli ECO5 100 30 648 0.43 MBI 11G13CN P. vulgaris SBPV1 3 3 >128 >128 2 MBI 11G13CN P.vulgaris SBPV1 3 3 >128 64 1.25 MBI 11G13CN S. aureus SBSA3 100 100 6464 2 MBI 11G13CN S. marcescens SBSM1 >100 >100 >128 >128 2

The MIC values of Mupirocin against strains of E. coli, S. aureus, P.aeruginosa are reduced by at least three fold in combination withindolicidin analogues at concentrations that are ≦½ MIC value of thepeptide alone.

Table 9 shows exemplary synergy data for combinations of indolicidinanalogues and Ciprofloxacin.

TABLE 9 Ciprofloxacin Peptide Ciprofloxacin Comb. Peptide Comb MIC MICMIC MIC Peptide Organism (μg/ml) (μg/ml) (μg/ml) (μg/ml) FIC MBI 11D18CNS. aureus SA14 16 8 8 4 1.00 MBI 11D18CN P. aeruginosa PA24 16 4 >128 160.31 MBI 11D18CN S. aureus SA10 32 32 2 2 2.00

The MIC values of Ciprofloxacin against strains of S. aureus and P.aeruginosa are reduced by at least two fold in combination withindolicidin analogues at concentrations that are ≦½ MIC value of thepeptide alone.

Example 5 Biochemical Characterization of Peptide Analogues

Solubility in Formulation Buffer

The primary factor affecting solubility of a peptide is its amino acidsequence. Polycationic peptides are preferably freely soluble in aqueoussolutions, especially under low pH conditions. However, in certainformulations, polycationic peptides may form an aggregate that isremoved in a filtration step. As peptide solutions for in vivo assaysare filtered prior to administration, the accuracy and reproducibilityof dosing levels following filtration are examined.

Peptides dissolved in formulations are filtered through a hydrophilic0.2 μm filter membrane and then analyzed for total peptide content usingreversed-phase HPLC. A 100% soluble standard for each concentration isprepared by dissolving the peptide in MilliQ water. Total peak area foreach condition is measured and compared with the peak area of thestandard in order to provide a relative recovery value for eachconcentration/formulation combination.

MBI 11CN was prepared in four different buffer systems (A, B, C, and C1)(Table 10, below) at 50, 100, 200 and 400 μg/ml peptide concentrations.With formulations A or B, both commonly used for salvation of peptidesand proteins, peptide was lost through filtration in a concentrationdependent manner (FIG. 4). Recovery only reached a maximum of 70% at aconcentration of 400 μg/ml. In contrast, peptides dissolved informulations C and C1 were fully recovered. Buffers containingpolyanionic ions appear to encourage aggregation, and it is likely thatthe aggregate takes the form of a matrix which is trapped by the filter.Monoanionic counterions are more suitable for the maintenance ofpeptides in a non-aggregated, soluble form, while the addition of othersolubilizing agents may further improve the formulation.

TABLE 10 Code Formulation Buffer A PBS 200 mM, pH 7.1 B Sodium Citrate100 mM, pH 5.2 C Sodium Acetate 200 mM, pH 4.6 C1 Sodium Acetate 200mM/0.5% Polysorbate 80, pH 4.6 D Sodium Acetate 100 mM/0.5% ActivatedPolysorbate 80, pH 7.5: Lyophilized/ReconstitutedSolubility in Broth

The solubility of peptide analogues is assessed in calcium and magnesiumsupplemented Mueller Hinton broth by visual inspection. The procedureemployed is that used for the broth dilution assay except that bacteriaare not added to the wells. The appearance of the solution in each wellis evaluated according to the scale: (a) clear, no precipitate, (b)light diffuse precipitate and (c) cloudy, heavy precipitate. Resultsshow that, for example, MBI 10CN is less soluble than MBI 11CN underthese conditions and that MBI 11BCN analogues are less soluble than MBI11ACN analogues.

Reversed Phase HPLC Analysis of Peptide Analogue Formulations

Reversed-phase HPLC, which provides an analytical method for peptidequantification, is used to examine peptides in two differentformulations. A 400 μg/mL solution of MBI 11CN prepared in formulationsC1 and D is analyzed by using a stepwise gradient to resolve freepeptide from other species. Standard chromatographic conditions are usedas follows:

-   -   Solvent A: 0.1% trifluoroacetic acid (TFA) in water    -   Solvent B: 0.1% TFA/95% acetonitrile in water    -   Media: POROSO® R2-20 (polystyrene divinylbenzene)

As shown in FIG. 5, MBI 11CN could be separated in two forms, as freepeptide in formulation C1, and as a principally formulation-complexpeptide in formulation D. This complex survives the separation protocolin gradients containing acetonitrile, which might be expected to disruptthe stability of the complex. A peak corresponding to a small amount(<10%) of free peptide is also observed in formulation D. If the shapeof the elution gradient is changed, the associated peptide elutes as abroad low peak, indicating that complexes of peptide in the formulationare heterogeneous.

Example 6 Structural Analysis of Indolicidin Variants Using CircularDichroism Spectroscopy

Circular dichroism (CD) is a spectroscopic technique that measuressecondary structures of peptides and proteins in solution, see forexample, R. W. Woody, (Methods in Enzymology, 246: 34, 1995). The CDspectra of α-helical peptides is most readily interpretable due to thecharacteristic double minima at 208 and 222 nm. For peptides with othersecondary structures however, interpretation of CD spectra is morecomplicated and less reliable. The CD data for peptides is used torelate solution structure to in vitro activity.

CD measurements of indolicidin analogues are performed in threedifferent aqueous environments, (1) 10 mM sodium phosphate buffer, pH7.2, (2) phosphate buffer and 40% (v/v) trifluoroethanol (TFE) and (3)phosphate buffer and large (100 nm diameter) unilamellar phospholipidvesicles (liposomes) (Table 11). The organic solvent TFE and theliposomes provide a hydrophobic environment intended to mimic thebacterial membrane where the peptides are presumed to adopt an activeconformation.

The results indicate that the peptides are primarily unordered inphosphate buffer (a negative minima at around 200 nm) with the exceptionof MBI 11F4CN, which displays an additional minima at 220 nm (seebelow). The presence of TFE induces β-turn structure in MBI 11 and MBI11G4CN, and increases α-helicity in MBI 11F4CN, although most of thepeptides remain unordered. In the presence of liposomes, peptides MBI11CN and MBI 11B7CN, which are unordered in TFE, display β-turnstructure (a negative minima at around 230 nm) (FIG. 6). Hence,liposomes appear to induce more ordered secondary structure than TFE.

A β-turn is the predominant secondary structure that appears in ahydrophobic environment, suggesting that it is the primary conformationin the active, membrane-associated form. In contrast, MBI 11F4CNdisplays increased α-helical conformation in the presence of TFE.Peptide MBI 11F4CN is also the most insoluble and hemolytic of thepeptides tested, suggesting that α-helical secondary structure mayintroduce unwanted properties in these analogues.

Additionally CD spectra are recorded for APS-modified peptides (Table11). The results show that these compounds have significant β-turnsecondary structure in phosphate buffer, which is only slightly alteredin TFE.

Again, the CD results suggest that a β-turn structure (i.e.membrane-associated) is the preferred active conformation among theindolicidin analogues tested.

TABLE 11 Phosphate buffer Conformation TFE Conformation Peptide min λmax λ in buffer min λ max λ in TFE MBI 10CN 201 — Unordered 203 ~219 Unordered MBI 11 199 — Unordered 202, 227 220 β-turn MBI 11ACN 199 —Unordered 203 219 Unordered MBI 11CN 200 — Unordered 200 — Unordered MBI11CNY1 200 — Unordered 200 — Unordered MBI 11B1CNW1 201 — Unordered 201— Unordered MBI 11B4ACN 200 — Unordered 200 — Unordered MBI 11B7CN 200 —Unordered 204, ~219 Unordered MBI 11B9ACN 200 — Unordered 200 —Unordered MBI 11B9CN 200 — Unordered 200 — Unordered MBI 11D1CN 200 —Unordered 204 — Unordered MBI 11E1CN 201 — Unordered 201 — Unordered MBI11E2CN 200 — Unordered 201 — Unordered MBI 11E3CN 202 226 ppII helix 200— Unordered MBI 11F3CN 199 228 ppII helix 202 — Unordered MBI 11F4CN202, 220 — Unordered 206, 222 — slight α-helix MBI 11G4CN 199, 221 —Unordered 201, 226 215 β-turn MBI 11G6ACN 200 — Unordered 199 —Unordered MBI 11G7ACN 200 — Unordered 202 221 Unordered

TABLE 12 APS-modified Phosphate buffer Conformation TFE Conformationpeptide min λ max λ in buffer min λ max λ in TFE MBI 11CN 202, 229 220β-turn 203 223 β-turn MBI 11BCN 200, 229 — β-turn 202 222 β-turn MBI11B7CN 202, 230 223 β-turn 199 230 β-turn MBI 11E3CN 202, 229 220 β-turn199 — β-turn MBI 11F3CN 205 — ppII helix 203 230 ppII helix

Example 7 Membrane Permeabilization Assays

Liposome Dye Release

A method for measuring the ability of peptides to permeabilizephospholipid bilayers is described (Parente et al., Biochemistry, 29,8720, 1990) Briefly, liposomes of a defined phospholipid composition areprepared in the presence of a fluorescent dye molecule. In this example,a dye pair consisting of the fluorescent molecule8-aminonapthalene-1,3,6-trisulfonic acid (ANTS) and its quenchermolecule p-xylene-bis-pyridinium bromide (DPX) are used. The mixture offree dye molecules, dye free liposomes, and liposomes containingencapsulated ANTS-DPX are separated by size exclusion chromatography. Inthe assay, the test peptide is incubated with the ANTS-DPX containingliposomes and the fluorescence due to ANTS release to the outside of theliposome is measured over time.

Using this assay, peptide activity, measured by dye release, is shown tobe extremely sensitive to the composition of the liposomes at manyliposome to peptide ratios (L/P) (FIG. 7). Specifically, addition ofcholesterol to liposomes composed of egg phosphotidylcholine (PC)virtually abolishes membrane permeabilizing activity of MBI 11CN, evenat very high lipid to peptide molar ratios (compare with egg PCliposomes containing no cholesterol). This in vitro selectivity maymimic that observed in vitro for bacterial cells in the presence ofmammalian cells.

In addition, there is a size limitation to the membrane disruptioninduced by MBI 11CN. ANTS/DPX can be replaced with fluoresceinisothiocyanate-labeled dextran (FD-4), molecular weight 4,400, in theegg PC liposomes. No increase in FD-4 fluorescence is detected uponincubation with MBI 11CN. These results indicate that MBI 11CN-mediatedmembrane disruption allows the release of the relatively smallerANTS/DPX molecules (˜400 Da), but not the bulkier FD-4 molecules.

E. coli ML-35 Inner Membrane Assay

An alternative method for measuring peptide-membrane interaction usesthe E. coli strain ML-35 (Lehrer et al., J. Clin. Invest., 84: 553,1989), which contains a chromosomal copy of the lacZ gene encodingβ-galactosidase and is permease deficient. This strain is used tomeasure the effect of peptide on the inner membrane through release ofβ-galactosidase into the periplasm. Release of β-galactosidase ismeasured by spectrophotometrically monitoring the hydrolysis of itssubstrate o-nitrophenol β-D-galactopyranoside (ONPG). The maximum rateof hydrolysis (V_(max)) is determined for aliquots of cells taken atvarious growth points.

A preliminary experiment to determine the concentration of peptiderequired for maximal activity against mid-log cells, diluted to 4×10⁷CFU/ml, yields a value of 50 μg/ml, which is used in all subsequentexperiments. Cells are grown in two different growth media, Terrificbroth (TB) and Luria broth (LB) and equivalent amounts of cells areassayed during their growth cycles. The resulting activity profile ofMBI 11B7CN is shown in FIG. 8. For cells grown in the enriched TB media,maximum activity occurs at early mid-log (140 min), whereas for cellsgrown in LB media, the maximum occurs at late mid-log (230 min).Additionally, only in LB, a dip in activity is observed at 140 min. Thisdrop in activity may be related to a transition in metabolism, such as arequirement for utilization of a new energy source due to depletion ofthe original source, which does not occur in the more enriched TB media.A consequence of a metabolism switch would be changes in the membranepotential.

To test whether membrane potential has an effect on peptide activity,the effect of disrupting the electrochemical gradient using thepotassium ionophore valinomycin is examined. Cells pre-incubated withvalinomycin are treated with peptide and for MBI 10CN and MBI 11CN ONPGhydrolysis diminished by approximately 50% compared to no pre-incubationwith valinomycin (FIG. 9). Another cationic peptide that is notsensitive to valinomycin is used as a positive control.

Further delineation of the factors influencing membrane permeabilizingactivity are tested. In an exemplary test, MBI 11B7CN is pre-incubatedwith isotonic HEPES/sucrose buffer containing either 150 mM sodiumchloride (NaCl) or 5 mM magnesium ions (Mg²⁺) and assayed as describedearlier. In FIG. 10, a significant inhibition is observed with eithersolution, suggesting involvement of electrostatic interactions in thepermeabilizing action of peptides.

Example 8 Erythtocyte Lysis by Indolicidin Analogues

A red blood cell (RBC) lysis assay is used to group peptides accordingto their ability to lyse RBC under standardized conditions compared withMBI 11CN and Gramicidin-S. Peptide samples and washed sheep RBC areprepared in isotonic saline with the final pH adjusted to between 6 and7. Peptide samples and RBC suspension are mixed together to yieldsolutions that are 1% (v/v) RBC and 5, 50 or 500 μg/ml peptide. Assaymixtures are incubated for 1 hour at 37° C. with constant shaking,centrifuged, and the supernatant is measured for absorbance at 540 nm,which detects released hemoglobin. The percentage of released hemoglobinis determined by comparison with a set of known standards lysed inwater. Each set of assays also includes MBI 11CN (500 μg/ml) andGramicidin-S (5 μg/ml) as “low lysis” and “high lysis” controls,respectively.

MBI-11B7CN-HCl, MBI-11F3CN-HCl and MBI-11F4CN-HCl are tested using thisprocedure and the results are presented in Table 13 below.

TABLE 13 % lysis at % lysis at % lysis at Peptide 5 μg/ml 50 μg/ml 500μg/ml MBI 11B7CN-HCl 4 13 46 MBI 11F3CN-HCl 1 6 17 MBI 11F4CN-HCl 4 3238 MBI 11CN-TFA N/D N/D 9 Gramicidin-S 30 N/D N/D N/D = not done

Peptides that at 5 μg/ml lyse RBC to an equal or greater extent thanGramicidin-S, the “high lysis” control, are considered to be highlylytic. Peptides that at 500 μg/ml lyse RBC to an equal to or lesserextent than MBI 11CN, the “low lysis” control, are considered to benon-lytic. The three analogues tested are all “moderately lytic” as theycause more lysis than MBI 11CN and less than Gramicidin-S. In additionone of the analogues, MBI-11F3CN-HCl, is significantly less lytic thanthe other two variants at all three concentrations tested.

Example 9 Production of Antibodies to Peptide Analogues

Multiple antigenic peptides (MAPs), which contain four or eight copiesof the target peptide linked to a small non-immunogenic peptidyl core,are prepared as immunogens. Alternatively, the target peptide isconjugated to bovine serum albumin (BSA) or ovalbumin. For example, MBI11CN and its seven amino acid N-terminal and C-terminal fragments areused as target peptide sequences. The immunogens are injectedsubcutaneously into rabbits using standard protocols (see, Harlow andLane, Antibodies: A Laboratory Mantial, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988). After repeated boosters (usuallymonthly), serum from a blood sample is tested in an ELISA against thetarget peptide. A positive result indicates the presence of antibodiesand further tests determine the specificity of the antibody binding tothe target peptide. Purified antibodies can then be isolated from thisserum and used in ELISAs to selectively identify and measure the amountof the target peptide in research and clinical samples.

Example 10 Pharmacology of Peptide Analogues in Plasma and Blood

The in vitro lifetime of free peptide analogues in plasma and in bloodis determined by measuring the amount of peptide present after setincubation times. Blood is collected from sheep, treated with ananticoagulant (not heparin) and, for plasma preparation, centrifuged toremove cells. Formulated peptide is added to either the plasma fractionor to whole blood and incubated. Following incubation, peptide isidentified and quantified directly by reversed phase HPLC. Extraction isnot required as the free peptide peak does not overlie any peaks fromblood or plasma.

A 1 mg/mL solution of MBI 11CN in formulations C1 and D is added tofreshly prepared sheep plasma at a final peptide concentration of 100μg/mL and incubated at 37° C. At various times, aliquots of plasma areremoved and analyzed for free peptide by reversed phase HPLC. From eachchromatogram, the area of the peak corresponding to free peptide isintegrated and plotted against time of incubation. As shown in FIG. 11,peptide levels diminish over time. Moreover, when administered informulation D, up to 50% of the peptide is immediately released fromformulation-peptide complex on addition to the blood. The decay curvefor free peptide yields an apparent half-life in blood of 90 minutes forboth formulation C1 and D. These results indicate that in sheep's bloodMBI 11CN is relatively resistant to plasma peptidases and proteases. Newpeaks that appeared during incubation may be breakdown products of thepeptide.

Peptide levels in plasma in vivo are measured after iv or ipadministration of 80-100% of the maximum tolerated dose of peptideanalogue in either formulation C1 or D. MBI 11CN in formulation C1 isinjected intravenously into the tail vein of CD1 ICRBR strain mice. Atvarious times post-injection, mice are anesthetized and blood is drawnby cardiac puncture. Blood from individual mice is centrifuged toseparate plasma from cells. Plasma is then analyzed by reversed phaseHPLC column. The resulting elution profiles are analyzed for freepeptide content by UV absorbance at 280 nm, and these data are convertedto concentrations in blood based upon a calibrated standard. Each datapoint represents the average blood level from two mice. In this assay,the detection limit is approximately 1 μg/ml, less than 3% of the doseadministered

The earliest time point at which peptide can be measured is threeminutes following injection, thus, the maximum observed concentration(in μg/ml) is extrapolated back to time zero (FIG. 12). The projectedinitial concentration corresponds well to the expected concentration ofbetween 35 and 45 μg/ml. Decay is rapid, however, and when the curve isfitted to the equation for exponential decay, free circulating peptideis calculated to have a half life of 2.1 minutes. Free circulatingpeptide was not detectable in the blood of mice that were injected withMBI 11CN in formulation D, suggesting that peptide is not released asquickly from the complex as in vitro.

In addition, MBI 11CN is also administered to CD1 ICRBR strain mice by asingle ip injection at an efficacious dose level of 40 mg/kg. Peptide isadministered in both formulations C1 and D to determine if peptidecomplexation has any effect on blood levels. At various times postinjection, mice are anesthetized and blood is drawn by cardiac puncture.Blood is collected and analyzed as for the iv injection.

MBI 11CN administered by this route demonstrated a quite differentpharmacologic profile (FIG. 13). In formulation C1, peptide entered theblood stream quickly, with a peak concentration of nearly 5 μg/ml after15 minutes, which declined to non-detectable levels after 60 minutes. Incontrast, peptide in formulation D is present at a level above 2 μg/mlfor approximately two hours. Therefore, formulation affects entry into,and maintenance of levels of peptide in the blood.

Example 11 Toxicity of Peptide Analogues In vivo

The acute, single dose toxicity of various indolicidin analogues istested in Swiss CD1 mice using various routes of administration. Inorder to determine the inherent toxicities of the peptide analogues inthe absence of any formulation/delivery vehicle effects, the peptidesare all administered in isotonic saline with the final pH between 6 and7.

Intraperitoneal route. Groups of 6 mice are injected with peptide dosesof between 80 and 5 mg/kg in 500 μl dose volumes. After peptideadministration, the mice are observed for a period of 5 days, at whichtime the dose causing 50% mortality (LD₅₀), the dose causing 90-100%mortality (LD₉₀₋₁₀₀) and maximum tolerated dose (MTD) levels aredetermined. The LD₅₀ values are calculated using the method of Reed andMuench (J. of Amer. Hyg. 27: 493-497, 1938). The results presented inTable 14 show that the LD₅₀ values for MBI 11CN and analogues range from21 to 52 mg/kg.

TABLE 14 Peptide LD₅₀ LD₉₀₋₁₀₀ MTD MBI 11CN 34 mg/kg 40 mg/kg 20 mg/kgMBI 11B7CN 52 mg/kg >80 mg/kg   30 mg/kg MBI 11E3CN 21 mg/kg 40 mg/kg<20 mg/kg   MBI 11F3CN 52 mg/kg 80 mg/kg 20 mg/kg

Intravenous route. Groups of 6 mice are injected with peptide doses of20, 16, 12, 8, 4 and 0 mg/kg in 100 μl volumes (4 ml/kg). Afteradministration, the mice are observed for a period of 5 days, at whichtime the LD₅₀, LD₉₀₋₁₀₀ and MTD levels are determined. The results fromthe IV toxicity testing of MBI 11CN and three analogues are shown inTable 15. The LD₅₀, LD₉₀₋₁₀₀ and MTD values range from 5.8 to 15 mg/kg,8 to 20 mg/kg and <4 to 12 mg/kg respectively.

TABLE 15 Peptide LD₅₀ LD₉₀₋₁₀₀ MTD MBI 11CN HCl 5.8 mg/kg 8.0 mg/kg <4mg/kg MBI 11B7CN HCl 7.5 mg/kg 16 mg/kg 4 mg/kg MBI 11F3CN HCl 10 mg/kg12 mg/kg 8 mg/kg MBI 11F4CN HCl 15 mg/kg 20 mg/kg 12 mg/kg

Subcutaneous route. The toxicity of MBI 11CN is also determined aftersubcutaneous (SC) administration. For SC toxicity testing, groups of 6mice are injected with peptide doses of 128, 96, 64, 32 and 0 mg/kg in300 μL dose volumes (12 mL/kg). After administration, the mice areobserved for a period of 5 days. None of the animals died at any of thedose levels within the 5 day observation period. Therefore, the LD₅₀,LD₉₀₋₁₀₀ and MTD are all taken to be greater than 128 mg/kg. Micereceiving higher dose levels showed symptoms similar to those seen afterIV injection suggesting that peptide entered the systemic circulation.These symptoms are reversible, disappearing in all mice by the secondday of observations.

The single dose toxicity of MBI 10CN and MBI 11CN in differentformulations is also examined in outbred ICR mice (Table 16).Intraperitoneal injection (groups of 2 mice) of MBI 10CN in formulationD show no toxicity up to 29 mg/kg and under the same conditions MBI 11CNshow no toxicity up to 40 mg/kg.

Intravenous injection (groups of 10 mice) of MBI 10CN in formulation Dshow a maximum tolerated dose (MTD) of 5.6 mg/kg (Table 16). Injectionof 11 mg/kg gave 40% toxicity and 22 mg/kg result in 100% toxicity.Intravenous injection of MBI 11CN in formulation C (lyophilized) show aMTD of 3.0 mg/kg. Injection at 6.1 mg/kg result in 10% toxicity and at12 mg/kg 100% toxicity.

TABLE 16 MTD Peptide Route # Animals Formulation (mg/kg) MBI 10CN ip 2formulation D >29 MBI 11CN ip 2 formulation D >40 MBI 10CN iv 10formulation D 5.6 MBI 11CN iv 10 formulation C 3.0 (lyophilized)

These results are obtained using peptide/buffer solutions that arelyophilized after preparation and reconstituted with water. If thepeptide solution is not lyophilized before injection, but usedimmediately after preparation, an increase in toxicity is seen, and themaximum tolerated dose can decrease by up to four-fold. For example, anintravenous injection of MBI 11CN as a non-lyophilized solution,formulation C1, at 1.5 mg/kg results in 20% toxicity and at 3.0 mg/kggave 100% toxicity. HPLC analyses of the non-lyophilized and lyophilizedformulations indicate that the MBI 11CN forms a complex withpolysorbate, and this complexation of the peptide reduces its toxicityin mice.

In addition, mice are multiply injected by an intravenous route with MBI11CN (Table 17). In one representative experiment, peptide administeredin 10 injections of 0.84 mg/kg at 5 minute intervals is not lethal.However, two injections of peptide at 4.1 mg/kg administered with a 10minute interval results in 60% mortality.

TABLE 17 # Dose Injec- Time Peptide Route Formulation Level* tionsInterval Result MBI 11CN iv formulation 0.84 10  5 min no D mortalityMBI 11CN iv formulation 4.1   2 10 min 66% D mortality *(mg/kg)

To assess the impact of dosing mice with peptide analogue, a series ofhistopathology investigations can be carried out. Groups of mice areadministered analogue at dose levels that are either at, or below theMTD, or above the MTD, a lethal dose. Multiple injections may be used tomimic possible treatment regimes. Groups of control mice are notinjected or injected with buffer only.

Following injection, mice are sacrificed at specified times and theirorgans immediately placed in a 10% balanced formalin solution. Mice thatdie as a result of the toxic effects of the analogue also have theirorgans preserved immediately. Tissue samples are taken and prepared asstained micro-sections on slides which are then examinedmicroscopically. Damage to tissues is assessed and this information canbe used to develop improved analogues, improved methods ofadministration or improved dosing regimes.

Example 12 In vivo Efficacy of Peptide Analogues

Analogues are tested for their ability to rescue mice from lethalbacterial infections. The animal model used is an intraperitoneal (ip)inoculation of mice with 10⁶-10⁸ Gram-positive organisms with subsequentadministration of peptide. The three pathogens investigated,methicillin-sensitive S. aureus (MSSA), methicillin-resistant S. aureus(MRSA), or S. epidermidis are injected ip into mice. For untreated mice,death occurs within 12-18 hours with MSSA and S. epidermis and within6-10 hours with MRSA.

Peptide is administered by two routes, intraperitoneally, at one hourpost-infection, or intravenously, with single or multiple doses given atvarious times pre- and post-infection.

MSSA infection. In a typical protocol, groups of 10 mice are infectedintraperitoneally with a LD₉₀₋₁₀₀ dose (5.2×10⁶ CFU/mouse) of MSSA(Smith, ATCC #19640) injected in brain-heart infusion containing 5%mucin. This strain of S. aureus is not resistant to any commonantibiotics. At 60 minutes post-infection, MBI 10CN or MBI 11CN, informulation D, is injected intraperitoneally at the stated dose levels.An injection of formulation alone serves as a negative control andadministration of ampicillin serves as a positive control. The survivalof the mice is monitored at 1, 2, 3 and 4 hrs post-infection and twicedaily thereafter for a total of 8 days.

As shown in FIG. 14, MBI 10CN is maximally active against MSSA (70-80%survival) at doses of 14.5 to 38.0 mg/kg, although 100% survival is notachieved. Below 14.5 mg/kg, there is clear dose-dependent survival. Atthese lower dose levels, there appears to be an animal-dependentthreshold, as the mice either die by day 2 or survive for the full eightday period. As seen in FIG. 15, MBI 11CN, on the other hand, rescued100% of the mice from MSSA infection at a dose level of 35.7 mg/kg, andwas therefore as effective as ampicillin. There was little or noactivity at any of the lower dose levels, which indicates that a minimumbloodstream peptide level must be achieved during the time that bacteriaare a danger to the host.

As shown above, blood levels of MBI 11CN can be sustained at a level ofgreater than 2 μ/ml for a two hour period inferring that this is higherthan the minimum level.

Additionally, eight variants based on the sequence of MBI 11CN aretested against MSSA using the experimental system described above.Peptides prepared in formulation D are administered at dose levelsranging from 12 to 24 mg/kg and the survival of the infected mice ismonitored for eight days (FIGS. 16-24). The percentage survival at theend of the observation period for each variant is summarized in Table18. As shown in the table, several of the variants showed efficacygreater than or equal to MBI 11CN under these conditions.

TABLE 18 % Survival 24 mg/kg 18 mg/kg 12 mg/kg 100 90 11B1CN, 11F3CN 8070 11E3CN 60 11B7CN 50 11CN 40 11G2CN 30 11B1CN 20 11G4CN 10 11CN,11B7CN, 11G2CN 11B8CN, 11F3CN 0 11A1CN 11A1CN, 11G2CN, 11CN, 11A1CN,11G4CN 11B1CN, 11B7CN, 11B8CN, 11F3CN, 11G4CN

S. epidermidis infection. Peptide analogues generally have lower MICvalues against S. epidermidis in vitro, therefore, lower blood peptidelevels might be more effective against infection.

In a typical protocol, groups of 10 mice are injected intraperitoneallywith an LD₉₀₋₁₀₀ dose (2.0×10⁸ CFU/mouse) of S. epidermidis (ATCC#12228) in brain-heart infusion broth containing 5% mucin. This strainof S. epidermidis is 90% lethal after 5 days. At 15 mins and 60 minspost-infection, various doses of MBI 11CN in formulation D are injectedintravenously via the tail vein. An injection of formulation only servesas the negative control and injection of gentamicin serves as thepositive control; both are injected at 60 minutes post-infection. Thesurvival of the mice is monitored at 1, 2, 3, 4, 6 and 8 hrspost-infection and twice daily thereafter for a total of 8 days.

As shown in FIGS. 25A and 25B, MBI 11CN prolongs the survival of themice. Efficacy is observed at all three dose levels with treatment 15minutes post-infection, however, there is less activity at 30 minutespost-infection and no significant effect at 60 minutes post-infection.Time of administration appears to be important in this model system,with a single injection of 6.1 mg/kg 15 minutes post-infection givingthe best survival rate.

MRSA infection. MRSA infection, while lethal in a short period of time,requires a much higher bacterial load than MSSA. In a typical protocol,groups of 10 mice are injected intraperitoneally with a LD₉₀₋₁₀₀ dose(4.2×10⁷ CFU/mouse) of MRSA (ATCC #33591) in brain-heart infusioncontaining 5% mucin. The treatment protocols are as follows, with thetreatment times relative to the time of infection:

0 mg/kg Formulation D alone (negative control), injected at 0 mins 5mg/kg Three 5.5 mg/kg injections at −5, +55, and +115 mins 1 mg/kg (2hr) Five 1.1 mg/kg injections at −5, +55, +115, +175 and +235 mins 1mg/kg (20 min) Five 1.1 mg/kg injections at −10, −5, 0, +5, and +10 minsVancomycin (positive control) injected at 0 mins

MBI 11CN is injected intravenously in the tail vein in formulation D.Survival of mice is recorded at 1, 2, 3, 4, 6, 8, 10, 12, 20, 24 and 30hrs post-infection and twice daily thereafter for a total of 8 days.There was no change in the number of surviving mice after 24 hrs (FIG.26).

The 1 mg/kg (20 min) treatment protocol, with injections 5 minutes apartcentered on the infection time, delayed the death of the mice to asignificant extent with one survivor remaining at the end of the study.The results presented in Table 19 suggest that a sufficiently high levelof MBI 11CN maintained over a longer time period would increase thenumber of mice surviving. The 5 mg/kg and 1 mg/kg (2 hr) results, wherethere is no improvement in survivability over the negative control,indicates that injections 1 hour apart, even at a higher level, are noteffective against MRSA.

TABLE 19 Time of Observation Percentage of Animals Surviving (Hourspost-infection) No Treatment Treatment 6  50% 70% 8 0 40% 10 0 30% 12 020%

Example 13 Activation of Polysorbate 80 by Ultraviolet Light

A solution of 2% (w/w) polysorbate 80 is prepared in water and placed ina suitable reaction vessel, such as a quartz cell. Other containers thatare UV translucent or even opaque can be used if provision is made for aclear light path or an extended reaction time. In addition, the vesselshould allow the exchange of air but minimize evaporation.

The solution is irradiated with ultraviolet light using a lamp emittingat 254 nm. Irradiation can also be performed using a lamp emitting at302 nm. The activation is complete in 1-14 days depending upon thecontainer, the depth of the solution, and air exchange rate. Thereaction is monitored by a reversed-phased HPLC assay, which measuresthe formation of APS-modified MBI 11CN when the light-activatedpolysorbate is reacted with MBI 11CN.

Some properties of activated polysorbate are determined. Becauseperoxides are a known by-product of exposing ethers to UV light,peroxide formation is examined through the effect of reducing agents onthe activated polysorbate. As seen in FIG. 27A, activated polysorbatereadily reacts with MBI 11CN. Pre-treatment with 2-mercaptoethanol (FIG.27B), a mild reducing agent, eliminates detectable peroxides, but doesnot cause a loss of conjugate forming ability. Treatment with sodiumborohydride (FIG. 27C), eliminates peroxides and eventually eliminatesthe ability of activated polysorbate to modify peptides. Hydrolysis ofthe borohydride in water raises the pH and produces borate as ahydrolysis product. However, neither a pH change nor borate areresponsible.

These data indicate that peroxides are not involved in the modificationof peptides by activated polysorbate. Sodium borohydride should notaffect epoxides or esters in aqueous media, suggesting that the reactivegroup is an aldehyde or ketone. The presence of aldehydes in theactivated polysorbate is confirmed by using a formaldehyde test, whichis specific for aldehydes including aldehydes other than formaldehyde.

Furthermore, activated polysorbate is treated with2,4-dinitrophenylhydrazine (DNPH) in an attempt to capture the reactivespecies. Three DNPH-tagged components are purified and analyzed by massspectroscopy. These components are polysorbate-derived with molecularweights between 1000 and 1400. This indicates that low molecular weightaldehydes, such as formaldehyde or acetaldehyde, are involved.

Example 14 Formation of APS-Modified Peptides

APS-modified peptides are prepared either in solid phase or liquidphase. For solid phase preparation, 0.25 ml of 4 mg/ml of MBI 11CN isadded to 0.5 ml of 0.4 M Acetic acid-NaOH pH 4.6 followed by addition of0.25 ml of UV-activated polysorbate. The reaction mix is frozen byplacing it in a −80° C. freezer. After freezing, the reaction mix islyophilized overnight.

For preparing the conjugates in an aqueous phase, a sample of UVactivated polysorbate 80 is first adjusted to a pH of 7.5 by theaddition of 0.1M NaOH. This pH adjusted solution (0.5 ml) is added to1.0 ml of 100 mM sodium carbonate, pH 10.0, followed immediately by theaddition of 0.5 ml of 4 mg/ml of MBI 11CN. The reaction mixture isincubated at ambient temperature for 22 hours. The progress of thereaction is monitored by analysis at various time points using RP-HPLC(FIG. 28). In FIG. 28, peak 2 is unreacted peptide, peak 3 isAPS-modified peptide. Type 1 is the left-most of peak 3 and Type 2 isthe right-most of peak 3.

Table 20 summarizes data from several experiments. Unless otherwisenoted in table 20, the APS-modified peptides are prepared via thelyophilization method in 200 mM acetic acid-NaOH buffer, pH 4.6.

TABLE 20 COMPLEX SEQUENCE NAME TYPE 1 TYPE 2 ILKKWPWWPWRRKamide 11CN(SEQ ID NO: 30) Solid phase, pH 2.0 Yes Low Solid phase, pH 4.6 Yes YesSolid phase, pH 5.0 Yes Yes Solid phase, pH 6.0 Yes Yes Solid phase, pH8.3 Yes Yes Solution, pH 2.0 Trace Trace Solution, pH 10.0 Yes Yes-Slow(Ac)₄-ILKKWPWWPWRRKamide 11CN-Y1 No No (SEQ ID NO: 30)ILRRWPWWPWRRKamide 11B1CN Yes Lowered (SEQ ID NO: 38) ILRWPWWPWRRKamide11B7CN Yes Lowered (SEQ ID NO: 42) ILWPWWPWRRKamide 11B8CN Yes Lowered(SEQ ID NO: 44) ILRRWPWWPWRRRamide 11B9CN Yes Trace (SEQ ID NO: 25)ILKKWPWWPWKKKamide 11B10CN Yes Yes (SEQ ID NO: 45) ILKKWPWWPWRRkamide11E3CN Yes Yes (SEQ ID NO: 64) ILKKWVWWPWRRKamide 11F3CN Yes Yes (SEQ IDNO: 67) ILKKWPWWPWKamide 11G13CN Yes Yes (SEQ ID NO: 28)ILKKWPWWPWRamide 11G14CN Yes Trace (SEQ ID NO: 75)

The modification of amino groups is further analyzed by determining thenumber of primary amino groups lost during attachment. The unmodifiedand modified peptides are treated with 2,4,6-trinitrobenzenesulfonicacid (TNBS) (R. L. Lundblad in Techniques in Protein Modification andAnalysis pp. 151-154, 1995) (Table 21).

Briefly, a stock solution of MBI 11CN at 4 mg/ml and an equimolarsolution of APS-modified MBI 11CN are prepared. A 0.225 ml aliquot ofMBI 11CN or APS-modified MBI 11CN is mixed with 0.225 ml of 200 mMsodium phosphate buffer, pH 8.8. A 0.450 ml aliquot of 1% TNBS is addedto each sample, and the reaction is incubated at 37° C. for 30 minutes.The absorbance at 367 nm is measured, and the number of modified primaryamino groups per molecule is calculated using an extinction coefficientof 10,500 M⁻¹ cm⁻¹ for the trinitrophenyl (TNP) derivatives.

The primary amino group content of the parent peptide is then comparedto the corresponding APS-modified peptide. As shown below, the loss of asingle primary amino group occurs during formation of modified peptide.Peptides possessing a 3,4 lysine pair consistently give results that are1 residue lower than expected, which may reflect steric hindrance aftertitration of one member of the doublet.

TABLE 21 TNP/APS- modified PEPTIDE SEQUENCE TNP/PEPTIDE peptide CHANGEILKKWPWWPWRRKamide 2.71 1.64 1.07 (SEQ ID NO: 30) ILRRWPWWPWRRKamide1.82 0.72 1.10 (SEQ ID NO: 38) IlKKWPWWPWRRkamide 2.69 1.61 1.08 (SEQ IDNO: 30) ILKKWVWWPWRRKamide 2.62 1.56 1.06 (SEQ ID NO: 67)Stability of APS-modified Peptide Analogues

APS-modified peptides demonstrate a high degree of stability underconditions that promote the dissociation of ionic or hydrophobiccomplexes. APS-modified peptide in formulation D is prepared as 800μg/ml solutions in water, 0.9% saline, 8M urea, 8M guanidine-HCl, 67%1-propanol, 1M HCl and 1M NaOH and incubated for 1 hour at roomtemperature. Samples are analyzed for the presence of free peptide usingreversed phase HPLC and the following chromatographic conditions:

-   -   Solvent A: 0.1% trifluoroacetic acid (TFA) in water    -   Solvent B: 0.1% TFA/95% acetonitrile in water    -   Media: POROS R2-20 (polystyrene divinylbenzene)    -   Elution: 0% B for 5 column volumes        -   0-25% B in 3 column volumes        -   25% B for 10 column volumes        -   25-95% B in 3 column volumes        -   95% B for 10 column volumes

Under these conditions, free peptide elutes exclusively during the 25% Bstep and formulation-peptide complex during the 95% B step. None of thedissociating conditions mentioned above, with the exception of 1M NaOHin which some degradation is observed, are successful in liberating freepeptide from APS-modified peptide. Additional studies are carried outwith incubation at 55° C. or 85° C. for one hour. APS-modified peptideis equally stable at 55° C. and is only slightly less stable at 85° C.Some acid hydrolysis, indicated by the presence of novel peaks in theHPLC chromatogram, is observed with the 1M HCl sample incubated at 85°C. for one hour.

Example 15 Purification of APS-Modified MBI 11CN

A large scale preparation of APS-modified MBI 11CN is purified.Approximately 400 mg of MBI 11CN is APS-modified and dissolved in 20 mlof water. Unreacted MBI 11CN is removed by RP-HPLC. The solvent is thenevaporated from the APS-modified MBI 11CN pool, and the residue isdissolved in 10 ml methylene chloride. The modified peptide is thenprecipitated with 10 ml diethyl ether. After 5 min at ambienttemperature, the precipitate is collected by centrifugation at 5000×gfor 10 minutes. The pellet is washed with 5 ml of diethyl ether andagain collected by centrifugation at 5000×g for 10 minutes. Thesupernatants are pooled for analysis of unreacted polysorbateby-products. The precipitate is dissolved in 6 ml of water and thenflushed with nitrogen by bubbling for 30 minutes to remove residualether. The total yield from the starting MBI 11CN was 43%.

Example 16 Biological Assays Using APS-Modified Peptide

All biological assays that compare APS-modified peptides with unmodifiedpeptides are performed on an equimolar ratio. The concentration ofAPS-modified peptides can be determined by spectrophotometricmeasurement, which is used to normalize concentrations for biologicalassays. For example, a 1 mg/ml APS-modified MBI 11CN solution containsthe same amount of peptide as a 1 mg/ml MBI 11CN solution, thus allowingdirect comparison of toxicity and efficacy data.

APS-modified peptides are at least as potent as the parent peptides inin vitro assays performed as described herein. MIC values against grampositive bacteria are presented for several APS-modified peptides andcompared with the values obtained using the parent peptides (Table 22).The results indicate that the modified peptides are at least as potentin vitro as the parent peptides and may be more potent than the parentpeptides against E. faecalis strains.

TABLE 22 Corrected MIC (μg/ml) Organism Organism # Peptide APS-peptidePeptide A. calcoaceticus AC002 MBI 11B1CN 4 2 A. calcoaceticus AC002 MBI11B7CN 8 4 A. calcoaceticus AC002 MBI 11CN >64 4 A. calcoaceticus AC002MBI 11E3CN 8 2 A. calcoaceticus AC002 MBI 11F3CN 8 2 E. cloacae ECL007MBI 11B1CN 128 >128 E. cloacae ECL007 MBI 11B7CN 128 128 E. cloacaeECL007 MBI 11CN 64 >128 E. cloacae ECL007 MBI 11E3CN 128 >128 E. cloacaeECL007 MBI 11F3CN 128 >128 E. coli ECO005 MBI 11B1CN 16 8 E. coli ECO005MBI 11B7CN 64 8 E. coli ECO005 MBI 11CN 64 16 E. coli ECO005 MBI 11E3CN64 8 E. coli ECO005 MBI 11F3CN 128 16 E. faecalis EFS001 MBI 11B1CN 4 32E. faecalis EFS001 MBI 11B7CN 8 8 E. faecalis EFS001 MBI 11CN 8 32 E.faecalis EFS001 MBI 11E3CN 4 8 E. faecalis EFS001 MBI 11F3CN 8 32 E.faecalis EFS004 MBI 11B1CN 4 8 E. faecalis EFS004 MBI 11B7CN 8 8 E.faecalis EFS004 MBI 11CN 4 8 E. faecalis EFS004 MBI 11E3CN 4 2 E.faecalis EFS004 MBI 11F3CN 4 16 E. faecalis EFS008 MBI 11B1CN 8 32 E.faecalis EFS008 MBI 11B7CN 8 32 E. faecalis EFS008 MBI 11CN 64 64 E.faecalis EFS008 MBI 11E3CN 8 16 E. faecalis EFS008 MBI 11F3CN 4 128 K.pneumoniae KP001 MBI 11B1CN 32 128 K. pneumoniae KP001 MBI 11B7CN 64 16K. pneumoniae KP001 MBI 11CN 64 128 K. pneumoniae KP001 MBI 11E3CN 64 8K. pneumoniae KP001 MBI 11F3CN 128 64 P. aeruginosa PA004 MBI 11B1CN 128128 P. aeruginosa PA004 MBI 11B7CN 128 128 P. aeruginosa PA004 MBI 11CN64 >128 P. aeruginosa PA004 MBI 11E3CN 128 128 P. aeruginosa PA004 MBI11F3CN 128 128 S. aureus SA010 MBI 11B1CN 4 1 S. aureus SA010 MBI 11B7CN4 1 S. aureus SA010 MBI 11CN 4 2 S. aureus SA010 MBI 11E3CN 2 1 S.aureus SA010 MBI 11F3CN 4 2 S. aureus SA011 MBI 11B1CN 16 4 S. aureusSA011 MBI 11B7CN 16 4 S. aureus SA011 MBI 11CN 16 8 S. aureus SA011 MBI11E3CN 16 4 S. aureus SA011 MBI 11F3CN 16 8 S. aureus SA014 MBI 11B1CN 48 S. aureus SA014 MBI 11B7CN 8 4 S. aureus SA014 MBI 11CN 8 16 S. aureusSA014 MBI 11E3CN 4 4 S. aureus SA014 MBI 11F3CN 8 8 S. aureus SA018 MBI11B1CN 32 16 S. aureus SA018 MBI 11B7CN 32 16 S. aureus SA018 MBI 11CN64 64 S. aureus SA018 MBI 11E3CN 32 16 S. aureus SA018 MBI 11F3CN 64 16S. aureus SA025 MBI 11B1CN 4 1 S. aureus SA025 MBI 11B7CN 2 1 S. aureusSA025 MBI 11CN 2 4 S. aureus SA025 MBI 11E3CN 2 1 S. aureus SA025 MBI11F3CN 4 2 S. aureus SA093 MBI 11B1CN 2 1 S. aureus SA093 MBI 11B7CN 2 1S. aureus SA093 MBI 11CN 2 2 S. aureus SA093 MBI 11E3CN 2 1 S. aureusSA093 MBI 11F3CN 2 1 S. maltophilia SMA002 MBI 11B1CN 64 128 S.maltophilia SMA002 MBI 11B7CN 128 32 S. maltophilia SMA002 MBI 11CN >64128 S. maltophilia SMA002 MBI 11E3CN 128 64 S. maltophilia SMA002 MBI11F3CN 128 64 S. marcescens SMS003 MBI 11B1CN 128 >128 S. marcescensSMS003 MBI 11B7CN 128 >128 S. marcescens SMS003 MBI 11CN 64 >128 S.marcescens SMS003 MBI 11E3CN 128 >128 S. marcescens SMS003 MBI 11F3CN128 >128

Toxicities of APS-modified MBI 11CN and unmodified MBI 11CN are examinedin Swiss CD-1 mice. Groups of 6 mice are injected iv with single dosesof 0.1 ml peptide in 0.9% saline. The dose levels used are 0, 3, 5, 8,10, and 13 mg/kg. Mice are monitored at 1, 3, and 6 hrs post-injectionfor the first day, then twice daily for 4 days. The survival data forMBI 11CN mice are presented in Table 23. For APS-modified MBI 11CN, 100%of the mice survived at all doses, including the maximal dose of 13mg/kg.

TABLE 23 Peptide administered Cumulative No. Cumulative No. (mg/kg) No.Dead/Total Dead Surviving Dead/Total % Dead 13 6/6 18 0 18/18 100 10 6/612 0 12/12 100 8 6/6 6 0 6/6 100 5 0/6 0 6 0/6 0 3 0/6 0 12 0/12 0 0 0/60 18 0/18 0

As summarized below, the LD₅₀ for MBI 11CN is 7 mg/kg (Table 24), withall subjects dying at a dose of 8 mg/ml. The highest dose of MBI 11CNgiving 100% survival was 5 mg/kg. The data show that APS-modifiedpeptides are significantly less toxic than the parent peptides.

TABLE 24 Test Peptide LD₅₀ LD₉₀₋₁₀₀ MTD MBI-11CN-TFA  7 mg/kg  8 mg/kg 5 mg/kg APS-MBI-11CN >13 mg/kg* >13 mg/kg* >13 mg/kg* *could not becalculated with available data.

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. Accordingly, the invention is not limited exceptas by the appended claims.

1. An indolicidin analogue of up to 35 amino acids that comprises one ofthe following sequences: Ile Leu Lys Lys Trp Pro Trp Pro Trp Arg Arg Lys(SEQ ID NO: 33); Ile Leu Lys Lys Tyr Pro Trp Tyr Pro Trp Arg Arg Lys(SEQ ID NO: 34); Ile Leu Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ IDNO: 40); Ile Leu Trp Pro Trp Trp Pro Arg Arg Lys (SEQ ID NO: 44); IleLeu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Ile Met Ile Leu Lys Lys AlaGly Ser (SEQ ID NO: 46); Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg LysMet Ile Leu Lys Lys Ala Gly Ser (SEQ ID NO: 47); Ile Leu Arg Trp Pro TrpTrp Pro Trp Arg Arg Lys Asp Met Ile Leu Lys Lys Ala Gly Ser (SEQ ID NO:48); Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 52);Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Met Ile Leu Lys Lys AlaGly Ser (SEQ ID NO: 55); Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg ArgIle Met Ile Leu Lys Lys Ala Gly Ser (SEQ ID NO: 56); Ile Leu Lys Lys TrpPro Trp Trp Pro Trp Arg Arg Met (SEQ ID NO: 58); Ile Leu Lys Lys Trp ProTrp Trp Pro Trp Arg Arg Ile Met (SEQ ID NO: 59); Ile Leu Lys Lys Trp TrpTrp Pro Trp Arg Lys (SEQ ID NO: 60); Ile Leu Lys Lys Trp Pro Trp Trp TrpArg Lys (SEQ ID NO: 61); Ile Leu Lys Lys Trp Val Trp Trp Val Trp Arg ArgLys (SEQ ID NO: 65); Ile Leu Lys Lys Trp Pro Trp Trp Val Trp Arg Arg Lys(SEQ ID NO:66); Ile Leu Lys Lys Trp Val Trp Trp Pro Trp Arg Arg Lys (SEQID NO: 67); Ile Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO:69); Ile Leu Lys Lys Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 70);Ile Leu Lys Lys Trp Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 71); Ile LeuLys Lys Trp Pro Trp Trp Trp Arg Arg Lys (SEQ ID NO: 72); Ile Leu Lys LysTrp Pro Trp Trp Pro Arg Arg Lys (SEQ ID NO: 73); Ala Leu Arg Trp Pro TrpTrp Pro Trp Arg Arg Lys (SEQ ID NO: 76); Ile Ala Arg Trp Pro Trp Trp ProTrp Arg Arg Lys (SEQ ID NO: 77); Ile Leu Ala Trp Pro Trp Trp Pro Trp ArgArg Lys (SEQ ID NO: 78); Ile Leu Arg Ala Pro Trp Trp Pro Trp Arg Arg Lys(SEQ ID NO: 79); Ile Leu Arg Trp Ala Trp Trp Pro Trp Arg Arg Lys (SEQ IDNO: 80); Ile Leu Arg Trp Pro Ala Trp Pro Trp Arg Arg Lys (SEQ ID NO:81); or Ile Leu Arg Trp Pro Trp Ala Pro Trp Arg Arg Lys (SEQ ID NO: 82).2. The indolicidin analogue according to claim 1 wherein the analoguehas at least one amino acid altered to a corresponding D-amino acid. 3.The indolicidin analogue according to claim 2 wherein the N-terminaland/or C-terminal amino acid is a D-amino acid.
 4. The indolicidinanalogue according to claim 1 wherein the analogue is acetylated at theN-terminal amino acid.
 5. The indolicidin analogue according to claim 1wherein the analogue is amidated at the C-terminal amino acid.
 6. Theindolicidin analogue according to claim 1 wherein the analogue isesterified at the C-terminal amino acid.
 7. The indolicidin analogueaccording to claim 1 wherein the analogue is modified by incorporationof homoserine/homoserine lactone at the C-terminal amino acid.
 8. Anindolicidin analogue the amino acid sequence of which consists of IleLeu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 42).
 9. Theindolicidin analogue of claim 8, wherein the analogue has at least oneamino acid altered to a corresponding D-amino acid.
 10. The indolicidinanalogue of claim 9, wherein the N-terminal and/or C-terminal amino acidis a D-amino acid.
 11. A pharmaceutical composition comprising aphysiologically acceptable buffer and an indolicidin analogue the aminoacid sequence of which consists of Ile Leu Arg Trp Pro Trp Trp Pro TrpArg Arg Lys (SEQ ID NO: 42).
 12. A pharmaceutical composition comprisinga physiologically acceptable buffer and at least one indolicidinanalogue of up to 35 amino acids that comprises one of the followingsequences: Ile Leu Lys Lys Trp Pro Trp Pro Trp Arg Arg Lys (SEQ ID NO:33); Ile Leu Lys Lys Tyr Pro Trp Tyr Pro Trp Arg Arg Lys (SEQ ID NO:34); Ile Leu Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 40);Ile Leu Trp Pro Trp Trp Pro Arg Arg Lys (SEQ ID NO: 44); Ile Leu Arg TrpPro Trp Trp Pro Trp Arg Arg Lys Ile Met Ile Leu Lys Lys Ala Gly Ser (SEQID NO: 46); Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys Met Ile LeuLys Lys Ala Gly Ser (SEQ ID NO: 47); Ile Leu Arg Trp Pro Trp Trp Pro TrpArg Arg Lys Asp Met Ile Leu Lys Lys Ala Gly Ser (SEQ ID NO: 48); Leu LysLys Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 52); Ile Leu Lys LysTrp Pro Trp Trp Pro Trp Arg Arg Met Ile Leu Lys Lys Ala Gly Ser (SEQ IDNO: 55); Ile Leu Lys Lys Trp Pro Trp Trp Pro Trp Arg Arg Ile Met Ile LeuLys Lys Ala Gly Ser (SEQ ID NO: 56); Ile Leu Lys Lys Trp Pro Trp Trp ProTrp Arg Arg Met (SEQ ID NO: 58); Ile Leu Lys Lys Trp Pro Trp Trp Pro TrpArg Arg Ile Met (SEQ ID NO: 59); Ile Leu Lys Lys Trp Trp Trp Pro Trp ArgLys (SEQ ID NO: 60); Ile Leu Lys Lys Trp Pro Trp Trp Trp Arg Lys (SEQ IDNO: 61); Ile Leu Lys Lys Trp Val Trp Trp Val Trp Arg Arg Lys (SEQ ID NO:65); Ile Leu Lys Lys Trp Pro Trp Trp Val Trp Arg Arg Lys (SEQ ID NO:66);Ile Leu Lys Lys Trp Val Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 67); IleLys Lys Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 69); Ile Leu LysLys Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 70); Ile Leu Lys Lys TrpTrp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 71); Ile Leu Lys Lys Trp Pro TrpTrp Trp Arg Arg Lys (SEQ ID NO: 72); Ile Leu Lys Lys Trp Pro Trp Trp ProArg Arg Lys (SEQ ID NO: 73); Ala Leu Arg Trp Pro Trp Trp Pro Trp Arg ArgLys (SEQ ID NO: 76); Ile Ala Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys(SEQ ID NO: 77); Ile Leu Ala Trp Pro Trp Trp Pro Trp Arg Arg Lys (SEQ IDNO: 78); Ile Leu Arg Ala Pro Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO:79); Ile Leu Arg Trp Ala Trp Trp Pro Trp Arg Arg Lys (SEQ ID NO: 80);Ile Leu Arg Trp Pro Ala Trp Pro Trp Arg Arg Lys (SEQ ID NO: 81); or IleLeu Arg Trp Pro Trp Ala Pro Trp Arg Arg Lys (SEQ ID NO: 82).
 13. Thepharmaceutical composition according to claim 12, wherein said at leastone analogue has one or more amino acids altered to a correspondingD-amino acid.
 14. The pharmaceutical composition according to claim 13,wherein the N-terminal and/or C-terminal amino acid of said at least oneanalogue is a D-amino acid.
 15. The pharmaceutical composition accordingto claim 12, wherein said at least one analogue is acetylated at theN-terminal amino acid.
 16. The pharmaceutical composition according toclaim 12, wherein said at least one analogue is amidated at theC-terminal amino acid.
 17. The pharmaceutical composition according toclaim 12, wherein said at least one analogue is esterified at theC-terminal amino acid.
 18. The pharmaceutical composition according toclaim 12, wherein said at least one analogue is modified byincorporation of homoserine/homoserine lactone at the C-terminal aminoacid.
 19. The pharmaceutical composition according to claim 12, whereinthe composition is incorporated in a liposome or a slow-release vehicle.20. A method of treating a microbial infection, comprising administeringto a patient a therapeutically effective amount of a pharmaceuticalcomposition according to any one of claims 12-19.
 21. The method ofclaim 20, wherein the infection is due to a microorganism selected fromthe group consisting of a bacterium, a fungus, and a protozoa.
 22. Themethod of claim 20, wherein the microorganism is a bacterium, whereinthe bacterium is a Gram-negative or a Gram-positive bacterium.
 23. Themethod of claim 20, wherein the pharmaceutical composition isadministered by intravenous injection, intraperitoneal injection orimplantation, intramuscular injection or implantation, intrathecalinjection, subcutaneous injection or implantation, intradermalinjection, lavage, bladder wash-out, suppositories, pessaries, oralingestion, topical application, enteric application, inhalation, ornasal route.
 24. The method of claim 20, wherein the pharmaceuticalcomposition is administered by local application.
 25. The method ofclaim 20, wherein the method is for treating a microbial infectionassociated with a medical device.
 26. The method according to claim 20,wherein the pharmaceutical composition further comprises an antibiotic.27. A method of treating a microbial infection, comprising administeringto a patient a therapeutically effective amount of a pharmaceuticalcomposition comprising an indolicidin analogue of up to 35 amino acidscomprising the sequence of Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg ArgLys (SEQ ID NO: 42).
 28. The method of claim 27, wherein the amino acidsequence consists of Ile Leu Arg Trp Pro Trp Trp Pro Trp Arg Arg Lys(SEQ ID NO: 42).
 29. The method of claim 27 or claim 28, wherein thepharmaceutical composition further comprises a physiologicallyacceptable buffer.
 30. The method of claim 27 or claim 28, wherein thepharmaceutical composition is administered by intravenous injection,intraperitoneal injection or implantation, intramuscular injection orimplantation, intrathecal injection, subcutaneous injection orimplantation, intradermal injection, lavage, bladder wash-out,suppositories, pessaries, oral ingestion, topical application, entericapplication, inhalation, or nasal route.
 31. The method of claim 27 orclaim 28, wherein the pharmaceutical composition is administered bylocal application.
 32. The method of claim 27 or claim 28, wherein themethod is for treating a microbial infection associated with a medicaldevice.
 33. The method of claim 27 or claim 28, wherein thepharmaceutical composition further comprises an antibiotic.
 34. Themethod of claim 33, wherein said antibiotic is minocycline.
 35. Themethod of claim 33, wherein said antibiotic is rifampin.
 36. The methodof claim 33, wherein said antibiotic is cefazolin.
 37. The method ofclaim 33, wherein said antibiotic is vancomycin.
 38. The method of claim33, wherein said antibiotic is teicoplanin.
 39. The method of claim 33,wherein said antibiotic is a beta lactam.
 40. The method of claim 27 orclaim 28, wherein the infection is due to a microorganism selected fromthe group consisting of a bacterium, a fungus, and a protozoa.
 41. Themethod of claim 40, wherein the microorganism is a Gram-negative or aGram-positive bacterium.
 42. The method of claim 40, wherein the fungusis a pathogenic yeast.
 43. The method of claim 40, wherein the fungus isa mold that causes wound infections.
 44. The method of claim 41, whereinsaid Gram-negative bacterium is selected from the group consisting ofEnterobacter sp., E. coli and Acinetobacter sp.
 45. The method of claim41, wherein said Gram-positive bacterium is selected from the groupconsisting of S. aureus, coagulase negative staphylococci, S.epidermidis, S. pneumoniae, and Viridans Streptococci.